Introduction

±3,4-Methylenedioxymethamphetamine (MDMA) is a widely used recreational drug that shows substantial promise as a psychotherapeutic agent (Feduccia et al. 2018; Sessa and Nutt 2015; UNODC 2020). MDMA targets various brain receptors and transporters with marked and preferential effects on the serotonergic system; it increases extracellular levels of serotonin (5-HT), norepinephrine (NE), and dopamine (DA) by reversing their transporters (SERT, NET, and DAT) and also exhibits some affinity for 5-HT, DA, muscarinic, histamine, and adrenergic receptors (Battaglia et al. 1988; Rothman et al. 2001; Rudnick and Wall 1992; Shulgin 1986; Torres et al. 2003). MDMA is classified chemically as a methamphetamine derivative, but behaviorally it is considered a stimulant-psychedelic by its detractors and an empathogen-entactogen by its proponents (Liechti 2015; Nichols 1986). It is these latter behavioral effects—increased empathy, trust, extroversion, and sociality (collectively referred to here as “prosocial”)—that distinguish MDMA from psychostimulants and hallucinogens and are of particular interest (Bershad et al. 2016; Dolder et al. 2018; Holze et al. 2020; Hysek et al. 2014; Kamilar-Britt and Bedi 2015; Liechti 2015; Nichols 1986; Schmid et al. 2014). Given these prosocial effects, MDMA has the potential to enhance the effectiveness of psychotherapy for psychiatric conditions such as social anxiety and autism spectrum disorders (Danforth et al. 2018) or even improve social behavior as a stand-alone treatment (Heifets and Malenka 2016). Recent phase 2 and 3 clinical studies also reveal that MDMA-assisted psychotherapy is an effective therapeutic for treatment-resistant post-traumatic stress disorder (Bouso et al. 2008; Mitchell et al. 2021; Mithoefer et al. 2011, 2013, 2018; Oehen et al. 2013; Ot’alora et al. 2018) that may outperform approved pharmacotherapies (i.e., the selective serotonin reuptake inhibitors paroxetine and sertraline) in terms of efficacy (Feduccia et al. 2019).

Despite MDMA’s apparent therapeutic promise, there is some concern regarding its behavioral toxicity (Schenk and Newcombe 2018), such as the potential to elicit memory impairments, addiction, and depressed mood. Moreover, it has been widely noted that a major hindrance to the development of psychedelics for therapeutic use is the lack of dose response data (for example, see Sellers et al. 2018). This data is critical to determine which doses of a therapeutic drug may be safe and effective. For example, psychostimulants (e.g., amphetamine, methylphenidate, cocaine, modafinil) are highly effective cognitive enhancers at ultra-low and low doses but can be addictive and cognitively impairing at high doses (for review, see Wood et al. 2014). We previously explored the role of dose in the cognitive effects of MDMA in a systematic review of existing literature (Pantoni and Anagnostaras 2019) and found no evidence in animals that MDMA impairs memory at low doses (< 3 mg/kg) but mixed results regarding cognitive effects at high doses (≥ 3 mg/kg). Since this review comprised mostly of single-dose studies and an assortment of methodologies, an empirical dose-ranging study on this topic is warranted. The current study aims to evaluate the conclusion from our systematic review that 3 mg/kg may be the threshold for MDMA-induced amnesia, and to further understand the dose-effect relationship of MDMA on behavioral assays of memory, addiction, and depression.

We have generally argued that doses should be scaled between animals and humans directly by body weight unless specific evidence (e.g., actual exposure data) justifies some specific kind of alternative scaling (see Carmack et al. 2014; Pantoni and Anagnostaras 2019, and Wood et al. 2014). Low-dose MDMA (about 1 to 2 mg/kg) produces equivalent increases in plasma drug concentration and monoamine release in humans (oral administration) and rodents (parenteral administration) (Baumann et al. 2007; Green et al. 2012), but time of peak drug exposure is shorter in rodents (10 to 45 min; Baumann et al. 2009) than in humans (about 145 min; Kolbrich et al. 2008). This data justifies temporal scaling but not dose scaling between rodent and human MDMA studies (for further discussion, see Pantoni and Anagnostaras 2019). Here, we systematically examine the effects of 0.01 to 10 mg/kg MDMA on Pavlovian fear conditioning; behavioral sensitization, conditioned place preference, and conditioned responding; and the Porsolt forced swim test in mice. This range captures doses from one-tenth to ten times those used in recent clinical studies (approximately 1–2 mg/kg MDMA; Bouso et al. 2008; Danforth et al. 2018; Mithoefer et al. 2011, 2013, 2018; Oehen et al. 2013; Ot’alora et al. 2018).

Pavlovian fear conditioning is a simple and efficient tool for modeling drug effects on learning and memory in rodents (Anagnostaras et al. 2000, 2010; Carmack et al. 2014; Maren 2001). In this task, an initially neutral conditioned stimulus (CS; e.g., a tone or an environmental context) is paired with an aversive unconditioned stimulus (US; e.g., a footshock). When learning occurs as a result of this pairing, either CS alone will elicit a conditioned response (CR; e.g., fear). In rodents, fear memory is typically quantified by measuring freezing behavior in response to a CS. Both context and tone fear memory are amygdala-dependent while contextual fear memory is also hippocampus-dependent (Anagnostaras et al. 1999, 2000, 2001, 2010; Gale et al. 2004; Maren et al. 1998). Psychostimulants modulate fear learning and memory dose-dependently: they enhance long-term memory at low, clinically relevant doses (0.005–0.05 mg/kg d-amphetamine; 0.01 and 1 mg/kg methylphenidate; 0.1 mg/kg cocaine; 0.75 mg/kg modafinil) but impair long-term memory at high, abused doses (4 and 8 mg/kg d-amphetamine; 10 mg/kg methylphenidate; 15 mg/kg cocaine; 75 mg/kg modafinil) (Carmack et al. 2014; Shuman et al. 2009; Wood and Anagnostaras 2009; Wood et al. 2007). Citalopram, a highly selective serotonin reuptake inhibitor, also impairs fear memory at high doses (10 mg/kg) but has no effect at low doses (0.01–1 mg/kg) (Carmack et al. 2014). Additional evidence suggests that psychostimulant-induced memory enhancement requires the combination of both DAT and NET inhibition (see Carmack et al. 2014; Pantoni et al. 2020).

Behavioral sensitization, conditioned place preference, and conditioned responding are behaviors that reflect the addictive potential^Footnote1 of a drug (Anagnostaras and Robinson 1996; Anagnostaras et al. 2002; Carmack et al. 2017; Robinson and Berridge 1993, 2003, 2008). Behavioral sensitization is a progressive increase in response following repeated administration of a drug and reflects dopamine system hyperactivation. Conditioned place preference is the preference for a context that has been paired with a drug and models the rewarding effects of a drug, as well as drug seeking. Conditioned responding after repeated environment-drug (CS-US) pairings is a drug-like CR to a drug-paired context and models associative learning thought to elicit craving. The effects of psychostimulants on these behaviors are also dose-dependent: low, memory-enhancing doses (0.005 mg/kg d-amphetamine; 1 mg/kg methylphenidate; 0.15 mg/kg cocaine; 0.75 mg/kg modafinil) show no evidence of sensitization or place preference while high, memory-impairing doses (1.5 mg/kg d-amphetamine; 10 mg/kg methylphenidate; 15 mg/kg cocaine; 75 mg/kg modafinil) show evidence of a high addictive potential (Carmack et al. 2014; Shuman et al. 2012). The action of high-dose psychostimulants at DAT and the ensuing increase in extracellular DA levels are largely responsible for the addictive potential of psychostimulants (Koob and Volkow 2010; Volkow et al. 1999, 2002); however, evidence suggests that drugs with weak activity at DAT (e.g., low affinity such as bupropion, or low dose such as Adderall) are not likely to produce addiction (Carmack et al. 2014; Pantoni et al. 2020).

The forced swim test is a model of depressive-like behavior and is used to screen for antidepressant drugs in rodents (Porsolt et al. 1977). In this test, animals are placed into a tank filled with water and time spent mobile (i.e., animal is active as it attempts to escape the stressful environment) versus immobile (i.e., “behavioral despair,” animal is passive as it loses hope to escape the stressful environment) is measured. Common antidepressants decrease immobility behavior in the forced swim test (Cryan et al. 2005a; Petit-Demouliere et al. 2005).

Published studies of MDMA’s effects on these behavioral tasks are primarily limited to high-dose experiments; low-dose and dose-ranging studies are lacking. On related contextual fear conditioning paradigms, rodents treated with 10 or 20 mg/kg then tested off-drug exhibited no effects (Shortall et al. 2013) or fear memory impairments (Johansson et al. 2015), respectively. On the forced swim test, rodents treated with 4 to 20 mg/kg then tested off-drug exhibited decreases (Majumder et al. 2011), increases (McGregor et al. 2003; Renoir et al. 2008; Shih et al. 2016, 2019; Thompson et al. 2004), or no changes (Abad et al. 2014; Clemens et al. 2005, 2007; Durkin et al. 2008; Ho et al. 2004) in depressive-like behavior. MDMA reliably produces behavioral sensitization in rodents treated with doses of 2 to 40 mg/kg (for example, see Åberg et al. 2007; Itzhak et al. 2003; Kalivas et al. 1998; McCreary et al. 1999; Spanos and Yamamoto 1989), and has been shown to produced conditioned responding in rodents treated with 5 mg/kg (Ciudad-Roberts et al. 2013; Gold and Koob 1989) but not in rodents treated with 3 mg/kg (McCreary et al. 1999) or 10 mg/kg (Anderson and Itzhak 2003; Varela et al. 2011). While the present study is the first to examine the effects of low-dose MDMA (≤ 1 mg/kg) on the aforementioned tasks, there are a few published dose-ranging studies on MDMA-induced conditioned place preference. Robledo et al. (2004) observed a significant conditioned place preference after repeated MDMA treatment in mice at a dose of 10 mg/kg but not at 0.3, 1, or 3.3 mg/kg. Similarly, Salzmann et al. (2003) observed a significant conditioned place preference after repeated MDMA treatment in mice at a dose of 9 mg/kg but not at 1 or 3 mg/kg. Still, in the present study, we analyze and compare the dose-effect relationship of MDMA across several behavioral tasks, utilizing the same methods as our previous psychostimulant studies (Carmack et al. 2014).

Methods

Subjects

A total of 184 hybrid C57BL/6Jx129S1/SvImJ (129B6; Jackson Laboratory, West Sacramento, CA, USA) male (n = 91) and female (n = 93) mice were used. This mouse strain was selected as per recommendations made at the Banbury Conference on Genetic Background in Mice to facilitate the comparison of results between experiments and among laboratories; this strain is often selected for excellent behavioral characteristics, and is a common genetic background of targeted mutations (Silva et al. 1997). Mice were weaned at 3 weeks of age and group housed (2–5 mice per same sex cage) with unrestricted access to food and water. The animal colony was maintained on a 14:10-h light/dark schedule and all testing occurred during the light phase. Mice were at least 10 weeks old and handled for 3 days (1 min/day) prior to testing. All 184 mice were used for fear conditioning; of these mice, 45 (24 males and 21 females) were used 6 weeks later for tests of behavioral sensitization, conditioned place preference, and conditioned responding, and 79 (33 males and 46 females) were used 8 weeks later for the forced swim test. Cages of mice were randomly re-assigned to these additional experiments. All animal care and experimental procedures were approved by the UCSD IACUC and compliant with the NRC Guide for the Care and Use of Laboratory Animals.

Drugs

3,4-MDMA HCl (CAS No. 64057-70-1; Cayman Chemical, Ann Arbor, MI, USA) was dissolved in 0.9 % physiological saline and given intraperitoneally (i.p.) in a volume of 10 mL/kg. A range of MDMA doses were selected: 0, 0.01, 0.05, 0.1, 0.5, 1, 3, and 10 mg/kg (salt weight).

Fear conditioning

The VideoFreeze system (Med Associates Inc., St. Albans, VT, USA) and fear conditioning protocol were used as described previously (Anagnostaras et al. 2000, 2010; Carmack et al. 2014; Pantoni et al. 2020; Shuman et al. 2009; Wood and Anagnostaras 2011). Four mice were tested concurrently in individual conditioning chambers (32 × 25 × 25 cm) that consisted of stainless-steel sidewalls and rod floors, white acrylic back walls, and clear polycarbonate front and top walls. Each chamber was transformed across multiple sensory dimensions to create two distinct contexts: a training context, which was used for training and context testing, and an alternate context, which was used for tone testing. For the training context, chambers were cleaned and scented with 7 % isopropanol, and illuminated with moderate (80 lx) white light and near-infrared light (980 nm). For the alternate context, chambers were outfitted with a black plastic, triangular teepee and white acrylic floors, cleaned and scented with a 5 % vinegar solution, and illuminated with only near-infrared light to create a dark environment. VideoFreeze software (Med Associates Inc.) used digital video to score freezing behavior and locomotor activity (Anagnostaras et al. 2010).

A total of 184 mice were randomly assigned to groups by dose of MDMA administered: 0 (n = 35), 0.01 (n = 20), 0.05 (n = 20), 0.1 (n = 30), 0.5 (n = 20), 1 (n = 20), 3 (n = 20), or 10 (n = 19) mg/kg. Groups were counterbalanced by sex and conditioning chamber. Mice were given an injection of MDMA or saline 30 min before a 10-min training session. A delay of 30 min was selected due to its temporal proximity to peak drug exposure, the first instance of locomotor activity (from pilot work in our lab), core temperature, and behavioral effects following intraperitoneal MDMA in mice (Fantegrossi et al. 2008; for review, see Pantoni and Anagnostaras 2019). Training began with a 3-min baseline period followed by a single tone-shock pairing, which consisted of a 30-s pure tone (2.8 kHz, 85 dBA) presented through a speaker in the chamber sidewall that co-terminated with a 2-s scrambled, AC constant current footshock (0.75 mA, RMS) delivered through the rod floors. Ninety seconds after the tone-shock pairing, mice underwent a 5-min post-shock test. Locomotor activity during the baseline period and during the footshock was used to measure on-drug baseline locomotion and shock reactivity, respectively, while freezing behavior during the post-shock test was used to measure on-drug short-term memory.

Seven days after training, mice were returned to the training context, off drug, for a 5-min context test. Freezing behavior during the test was used to measure long-term context memory. One day after context testing, mice were brought to the alternate context, off drug, for a 5-min tone test. Tone testing consisted of a 2-min baseline period, followed by the presentation of 3, 30-s tones identical to the training tone each separated by 30s. Freezing behavior during the tone presentations was used to measure long-term tone memory.

Behavioral sensitization, conditioned place preference, and conditioned responding

Eight mice were tested concurrently in individual place preference chambers (Med Associates Inc., St. Albans, VT, USA) as described previously (Carmack et al. 2013, 2014; Pantoni et al. 2020). Each chamber (43 × 43 × 31 cm) consisted of two sides—a drug-paired side and an unpaired side—separated by a black wall with a removable insert. The two sides were visually and tactilely distinct as they differed by flooring (stainless steel rods or wire mesh) and walls (white and decorated with stickers or undecorated clear polycarbonate). Chambers were counterbalanced by flooring and wall combinations and by paired versus unpaired side assignments. Each chamber was cleaned with 10 % glass cleaner (Zep Inc., Atlanta, GA, USA) between trials. Activity Monitor software (Med Associates Inc.) used the interruption of infrared beams to identify mouse position and score locomotion (distance), stereotypy (counts), and verticality (counts).Forty-five mice were randomly assigned to new groups by dose of MDMA administered: 0 (n = 12), 0.1 (n = 10), 1 (n = 11), or 10 (n = 12) mg/kg. These doses were selected to include doses that consistently had no effects in the fear conditioning experiment (0.1 and 1 mg/kg) and a dose that consistently impaired memory in the fear conditioning experiment (10 mg/kg). Groups were counterbalanced by sex and testing chamber. Mice were habituated to the testing chamber, off drug, for 30 min per side per day for 2 consecutive days prior to training (with the order of side placement counterbalanced). Four days after habituation, mice were trained every other day for a total of 7 days. On each training day, mice were injected with saline before being placed into the unpaired side for 15 min, then injected with MDMA before being placed into the paired side for 15 min. Locomotor, stereotyped, and vertical activity on the paired side was scored and behavioral sensitization was calculated as the difference between average activity on day 7 versus day 1.

Twenty-four hours after the last training day, mice were tested off drug for conditioned place preference. The inserts that previously separated the two sides of the chambers were removed. Mice were placed into the entryway between the two sides of the chamber (with the direction of entry counterbalanced) and allowed access to both sides for 15 min. Locomotor activity and time spent on each side was scored and place preference was calculated as the difference between responses on the paired side versus the unpaired side.

Forty-eight hours after the last training day, mice received two back-to-back challenge tests: one with saline and one with a high dose of MDMA (10 mg/kg). Mice were injected with saline and immediately placed into the paired side for 15 min and then removed and injected with 10 mg/kg MDMA and immediately returned to the paired side for 45 min. Locomotor, stereotyped, and vertical activity was scored to evaluate the presence of conditioned responding to the drug-paired side (saline challenge) and/or sensitized responding to the high dose of 10 mg/kg MDMA (high dose challenge). One mouse trained with 10 mg/kg MDMA died during the high dose challenge and its data was excluded from that test only.

Forced swim test

The forced swim test procedure was adapted from existing protocols (Can et al. 2012; Castagné et al. 2011; Porsolt et al. 2001; Yankelevitch-Yahav et al. 2015). Five mice were tested concurrently in individual cylindrical beaker-like glass tanks (10 cm diameter × 24 cm height) that were visually separated by white opaque acrylic dividers. Each tank was filled with water (24 ± 0.5 °C) to a depth of 15 cm. Mice were tested in moderate light (approximately 80 lx) and immobility was measured using an HD USB video camera and behavioral tracking software (ANY-Maze, Wood Dale, IL, USA; minimum immobility time = 2000 ms, immobility sensitivity = 75 %).

Seventy-nine mice were randomly assigned to new groups by dose of MDMA administered: 0 (n = 14), 0.1 (n = 13), 0.5 (n = 13), 1 (n = 13), 3 (n = 13), or 10 (n = 13) mg/kg. Groups were counterbalanced by sex and testing tank. Mice were given an injection of MDMA or saline 30 min before testing. Mice were placed into the water for a 6-min test and the time spent immobile was scored during the last 4 min to evaluate potential antidepressant effects (reduced immobility).

Statistical analyses

Data were analyzed using univariate or multivariate analyses of variance (ANOVAs/MANOVAs) to identify overall group differences. MANOVAs were used for repeated measures and within-subjects data (Fig. 2a, b, d, e, g, h). ANOVAs were used for individual pieces of between-subjects data (Figs. 1, 2c, f, i, 3, 4). Post hoc comparisons were performed following significant group differences using Fisher’s least significant difference (LSD) tests against the saline control group. Except for Supplementary Fig. 1, data from male and female mice were merged as we found no other statistically significant sex differences that meaningfully influenced these findings (p values > 0.05).

Fig. 1.

_{Effects of MDMA on fear learning and memory. a On-drug activity during the 3-min training baseline period and the 2-s footshock. Mice given 3 mg/kg MDMA showed increased baseline locomotion relative to saline controls. There were no group differences in shock reactivity. b Short-term memory as measured by percent freezing during the on-drug post-shock test. Mice given 10 mg/kg MDMA showed impaired short-term memory relative to saline controls. c Long-term context memory as measured by percent freezing during the off-drug context test, 1 week after training. Mice previously given 3 or 10 mg/kg MDMA showed impaired long-term context memory relative to saline controls. d Long-term tone memory as measured by percent freezing during the off-drug tone test, 1 day after context testing. Mice previously given 3 or 10 mg/kg MDMA showed impaired long-term tone memory relative to saline controls. Each point represents the mean ± 1 standard error. The gray bar indicates standard error range for the comparison saline control group. Asterisks identify significant comparisons against the saline control group using Fisher’s LSD (*P < 0.05, **P < 0.01, and ***P< 0.001)}

Fig. 2.

_{Effects of MDMA on behavioral sensitization. Mice were trained for 7 days and locomotion (a–c), stereotypy (d–f), and verticality (g–i) on the drug-paired side were measured. a, d, g Time course of activity on day 1 (left) and day 7 (right) of training. There were no group differences on day 1, but on day 7, mice receiving 10 mg/kg MDMA exhibited increased locomotion (a), stereotypy (d), and verticality (g) relative to saline controls. b, e, h Average activity on each of the seven days of training. Mice receiving 10 mg/kg MDMA exhibited increased locomotion (b), stereotypy (e), and verticality (h) relative to saline controls from day 3 to day 7. c, f, i Development of sensitization as measured by the difference in average activity on day 7 versus day 1. Mice receiving 10 mg/kg MDMA exhibited a greater increase in locomotion (c), stereotypy (f), and verticality (i) from day 1 to day 7 relative to saline controls. Asterisks identify significant comparisons against the saline control group at the same time point}

Fig. 3.

_{Effects of MDMA on conditioned place preference. Following 7 days of training, mice were tested off drug for place preference, which was measured by the difference in distance traveled (a) and time spent (b) on the drug-paired side versus the unpaired side. There were no significant group differences and none of the groups exhibited a significant preference for either side}

Fig. 4.

_{Effects of MDMA on conditioned and sensitized responding, and depressive-like behavior. a Following training and place preference testing, mice underwent saline (left) and high-dose MDMA (right) challenge tests on the paired side and locomotion was scored to evaluate conditioned and sensitized responding, respectively. Mice trained with 10 mg/kg MDMA showed increased locomotion relative to saline controls following both challenge injections. b A separate cohort of mice underwent a 6-min on-drug forced swim test and time spent immobile was measured during the last 4 min of testing. Mice given 3 or 10 mg/kg MDMA exhibited reduced immobility relative to saline controls}

Results

Fear conditioning

The effects of MDMA (0–10 mg/kg, i.p.) on fear learning and memory were examined using Pavlovian fear conditioning. Mice were trained on drug with a single tone-shock pairing. Freezing was scored during an on-drug post-shock test and 1 week later during an off-drug context test and an off-drug tone test to evaluate short- and long-term fear memory. MDMA weakly dose-dependently modulated locomotor activity during the training baseline period (F(7, 176) = 2.08, p = 0.05; Fig. 1a, lower line). Only mice given 3 mg/kg MDMA showed significantly increased baseline locomotion compared to saline controls (p = 0.001; all other p values > 0.07). The shock elicited a large activity burst unconditioned response that did not significantly differ between groups (F(7, 176) = 0.43, p = 0.88; Fig. 1a, upper line). MDMA dose-dependently modulated freezing during the on-drug post-shock (F(7, 176) = 5.24, p < 0.001; Fig. 1b), off-drug context (F(7, 176) = 7.17, p < 0.001; Fig. 1c), and off-drug tone (F(7, 176) = 3.98, p < 0.001; Fig. 1d) tests. Compared to saline controls, only mice given 10 mg/kg MDMA exhibited reduced freezing during the post-shock test (p < 0.001; all other p values > 0.05), and only mice previously given 3 or 10 mg/kg MDMA exhibited reduced freezing during the context (p values ≤ 0.03; all other p values > 0.1) and tone (p values ≤ 0.01; all other p values > 0.06) tests.

Behavioral sensitization, conditioned place preference, and conditioned responding

The effects of MDMA (0–10 mg/kg, i.p.) on addiction-related behaviors were examined using tests of behavioral sensitization, conditioned place preference, and conditioned responding. Mice were trained for 7 days in a two-sided chamber; on each day, mice were injected with saline and placed into the unpaired side and then injected with MDMA and placed into the paired side. Locomotor, stereotyped, and vertical activity on the drug-paired side was measured. Significant group differences in activity were not observed on day 1 (locomotion, F(3, 41) = 1.77, p = 0.17; stereotypy, F(3, 41) = 1.06, p = 0.38; verticality, F(3, 41) = 0.43, p = 0.74; Fig. 2a, d, g, left), but were observed on day 7 (locomotion, F(3, 41) = 11.85, p < 0.001; stereotypy, F(3, 41) = 7.54, p < 0.001; verticality, F(3, 41) = 6.82, p < 0.001; Fig. 2a, d, g, right). Compared to saline controls, only mice receiving 10 mg/kg MDMA showed significantly increased locomotor (p < 0.001; all other p values > 0.7), stereotyped (p = 0.001; all other p values > 0.3), and vertical (p < 0.001; all other p values > 0.6) activity on day 7.

There were also significant main effects of group (locomotion, F(3, 41) = 11.23, p < 0.001; stereotypy, F(3, 41) = 7.21, p < 0.001; verticality, F(3, 41) = 3.64, p = 0.02) and group-by-day interactions (locomotion, F(18, 246) = 3.6, p < 0.001; stereotypy, F(18, 246) = 2.51, p < 0.001; verticality, F(18, 246) = 3.51, p < 0.001) on average daily activity across the seven days of training (Fig. 2b, e, h). Compared to saline controls, only mice receiving 10 mg/kg MDMA showed significantly increased locomotor (p < 0.001; all other p values > 0.8), stereotyped (p = 0.001; all other p values > 0.5), and vertical (p = 0.007; all other p values > 0.6) activity, and these effects were observed on the last 5 days (locomotion, p values ≤ 0.002; stereotypy, p values ≤ 0.02; verticality, p values ≤ 0.01) but not the first 2 days of training (locomotion, p values > 0.07; stereotypy, p values > 0.09; verticality, p values > 0.1). Lastly, there were significant group differences in the development of sensitization as measured by the difference in average activity on day 7 versus day 1 (locomotion, F(3, 41) = 4.42, p = 0.009; stereotypy, F(3, 41) = 3.57, p = 0.02; verticality, F(3, 41) = 4.32, p = 0.01; Fig. 2c, f, i). Only mice receiving 10 mg/kg MDMA exhibited a significant increase in locomotor (p = 0.002; all other p values > 0.5), stereotyped (p = 0.03; all other p values > 0.3), and vertical (p = 0.04; all other p values > 0.1) activity from day 1 to day 7 when compared to saline controls.

Twenty-four hours after the last training day, mice were tested off drug for conditioned place preference. Mice were allowed free access to both sides and place preference was measured by the difference in distance traveled and time spent on the drug-paired side versus the unpaired side. There were no significant group differences in distance traveled (F(3, 41) = 0.48, p = 0.7; Fig. 3a) or time spent (F(3, 41) = 0.18, p = 0.91; Fig. 3b) between sides. Additionally, none of the groups exhibited place preference in locomotor activity (one sample two-tailed t-test against hypothesized μ = 0; t(9–11) values < 1, p values > 0.4) or time spent (t(9–11) values < 1.4, p values > 1).

Forty-eight hours after the last training day, mice were challenged with saline and then a high dose of MDMA (10 mg/kg) on the paired side. Locomotor, stereotyped, and vertical activity in response to the saline challenge and the high-dose challenge was scored to evaluate conditioned and sensitized responding, respectively. There were significant group differences in locomotion following the saline (F(3, 41) = 4.31, p = 0.01; Fig. 4a, left) and high-dose MDMA (F(3, 40) = 13.14, p < 0.001; Fig. 4b, right) challenges. Compared to saline controls, only mice trained with 10 mg/kg MDMA exhibited a CR as measured by increased locomotion following the saline challenge (p = 0.008; all other p values > 0.5) or sensitization as measured by increased locomotion following the high dose MDMA challenge (p < 0.001; all other p values > 0.5). The same pattern of effects was observed for stereotypy (group differences, p values ≤ 0.02; 10 mg/kg versus saline, p values ≤ 0.03) but not verticality (no group differences, p values > 0.2) (data not depicted).

Forced swim test

The effects of MDMA (0–10 mg/kg, i.p.) on depressive-like behavior were examined using the forced swim test. Mice underwent a 6-min on-drug test and time spent immobile was scored during the last 4 min of testing. MDMA dose-dependently modulated immobility (F(5, 73) = 13.13, p < 0.001; Fig. 4b). Only mice given 3 or 10 mg/kg MDMA exhibited reduced immobility relative to saline controls (p values < 0.001; all other p values > 0.5).

Discussion

The present study provides further evidence for the critical role of dose selection in the behavioral effects of MDMA. Specifically, we found that high doses of MDMA produced fear memory impairments (at 3 and 10 mg/kg), some evidence of an addictive potential (at 10 mg/kg), and antidepressant effects (at 3 and 10 mg/kg), while low doses of MDMA (≤ 1 mg/kg) did not. Frequent high-dose MDMA (≥ 3 mg/kg) should likely be avoided for its amnesic effects and addictive potential but low-dose MDMA, which has been administered in recent clinical studies (approximately 1–2 mg/kg MDMA; for review, see Feduccia et al. 2018), is likely safe in terms of the behaviors analyzed herein. It appears that MDMA has a narrow viable therapeutic window and lowering dose should remain an important consideration in clinical use.

Our earlier systematic review (Pantoni and Anagnostaras 2019) questioned concerns that therapeutic use of MDMA would cause memory problems, as there was no evidence from animal research that MDMA impairs cognition at low, clinically relevant doses (< 3 mg/kg) but results regarding higher doses (≥ 3 mg/kg) were mixed. The present dose-effect analysis provides further evidence that 3 mg/kg MDMA appears to be the threshold for memory impairments. Using a Pavlovian fear conditioning paradigm, 10 mg/kg MDMA impaired short-term memory (on drug), 3 and 10 mg/kg MDMA impaired long-term context and tone memory (off drug), and 0.01 to 1 mg/kg MDMA did not impair memory. These fear memory impairments were not confounded by effects on nociception, as demonstrated by lack of group differences in shock reactivity, nor by effects on locomotor activity, as the short-term memory-impairing dose of 10 mg/kg MDMA had no effect on baseline locomotion and the long-term memory tests were conducted off drug. Our data suggests that high-dose MDMA produces anterograde amnesia because short- and long-term fear memory impairments were comparable. Further investigation is required to isolate the nature of the observed fear memory impairments, which could be caused by failures to encode, consolidate, or retrieve the memory, associate the memory with an aversive outcome, discriminate the context or tone from other places the animal has been or sounds they have heard, or engage defensive behavior to diffuse predictors of shock (Gerlai 2001; Maren 2008).

We did not detect any MDMA-induced fear memory enhancements even though psychostimulants enhance memory at low, clinically relevant doses (Carmack et al. 2014; Shuman et al. 2009; Wood and Anagnostaras 2009; Wood et al. 2007) and there is sparse evidence that MDMA may sometimes enhance cognition (for review, see Pantoni and Anagnostaras 2019). Instead, MDMA produced dose-dependent effects that were similar to that of the selective serotonin reuptake inhibitor citalopram (i.e., no effects at low, clinically relevant doses; impairments at high doses; Carmack et al. 2014). It is possible that MDMA does not act strongly enough at DAT and NET to enhance fear memory or that drug action at SERT interferes with fear memory enhancement. Enhanced memory reconsolidation and fear extinction has been proposed as a potential therapeutic mechanism of MDMA-assisted psychotherapy for post-traumatic stress disorder (Feduccia and Mithoefer 2018). While we did not detect changes in fear learning at low, clinically relevant doses of MDMA, high-dose MDMA (7.8 mg/kg) has been reported to enhance fear memory extinction (Young et al. 2015, 2017), and further research should investigate the effects of low-dose MDMA on fear extinction.

The addictive potential of high-dose psychostimulants is reflected in their propensity to elicit dramatic locomotor stimulation, behavioral sensitization, conditioned place preference, and conditioned responding (Anagnostaras and Robinson 1996; Anagnostaras et al. 2002; Carmack et al. 2014, 2017; Robinson and Berridge 1993, 2003, 2008; Shuman et al. 2012). We found that treatment with low, clinically relevant doses of 0.01 and 1 mg/kg MDMA did not lead to any addiction-related behaviors, even when tested with the 10 mg/kg MDMA high-dose challenge. Treatment with a high, memory-impairing dose of 10 mg/kg MDMA did lead to behavioral sensitization and conditioned responding, but not acute locomotor stimulation or conditioned place preference. Other drug-pairing procedures similarly have been found to occasion behavioral sensitization or conditioned responding in the absence of conditioned place preference (Brown and Fibiger 1993; Carmack et al. 2013; Hemby et al. 1992; Rowlett et al. 1994; Seymour and Wagner 2008). Furthermore, we observed interesting sex differences in the effects of 10 mg/kg MDMA on acute locomotor activity, as only females showed increased locomotion starting on the first day of training (see Supplementary Results). This may be related to findings that females are more sensitive than males to the psychological effects of MDMA (for review, see Allott and Redman 2007 and Liechti et al. 2001). However, both sexes similarly developed sensitization.

Our observations are inconsistent with previous reports of MDMA-induced place preference in mice at high doses of about 10 m/kg (for example, see Robledo et al. 2004; Salzmann et al. 2003). It is possible that the weak place preference observed in our 10 mg/kg MDMA group was non-significant due to insufficient statistical power. It has also been reported that MDMA does not induce place preference in group-housed animals (Meyer et al. 2002). Still, even if this effect were significant, its magnitude (difference in time spent on the drug-paired versus unpaired side = 19.2 %) is far weaker than that observed in our previous psychostimulant studies (around 50 %; Carmack et al. 2014; Shuman et al. 2012). Other head-to-head comparisons of psychostimulants versus MDMA have similarly found that rodents treated with methamphetamine and methylphenidate (Mori et al. 2021) as well as amphetamine (Meyer et al. 2002) exhibit significant place preference but those treated with comparable doses of MDMA do not. Altogether, our findings suggest that repeated use of MDMA at high (but not low) doses may lead to compulsive drug taking and drug-cue elicited craving, although MDMA may be less rewarding and less likely to provoke drug seeking than psychostimulants and other drugs that induce strong conditioned place preference (for reviews, see Carmack et al. 2017 and Tzschentke 2007). Indeed, the level of locomotor activity seen after MDMA administration seems to suggest that even at high doses, it is only a very modest psychomotor stimulant.

There are opposing views regarding how MDMA modulates depressive symptoms—one view holds that MDMA exacerbates mood problems including depression (for review, see Morgan 2000), while the other holds that MDMA has antidepressant properties that are implicated in its therapeutic effects (Thal and Lommen 2018; Yazar-Klosinski and Mithoefer 2017). Recent clinical studies report both depression symptom improvement as a secondary outcome and depressed mood as a treatment-emergent adverse event following MDMA-assisted psychotherapy (Mithoefer et al. 2019). Using the forced swim test, we detected acute MDMA-induced antidepressant effects at high, memory-impairing doses of 3 and 10 mg/kg but not at lower doses of 0.1, 0.5, and 1 mg/kg. Drugs that induce acute locomotor stimulation can lead to a false positive result in the forced swim test (Porsolt et al. 1978). This is a common concern with psychostimulants; however, there is clinical data suggesting that psychostimulants do indeed alleviate depressive symptoms and thus the term “false positive” may be misleading (Candy et al. 2008; Castagné et al. 2011). It is unlikely that locomotor stimulation was responsible for decreased immobility in the present study as we found little evidence that a single dose of 3 or 10 mg/kg MDMA acutely stimulates locomotor activity when averaged across both sexes. Since we found no acute antidepressant effects at low, clinically relevant doses, it is possible that low-dose MDMA requires chronic administration to reduce depressive-like behavior as do low-dose selective serotonin reuptake inhibitor, norepinephrine reuptake inhibitor, tricyclic, and monoamine oxidase inhibitor antidepressants (Cryan et al. 2005a, b; Detke et al. 1997; Vázquez-Palacios et al. 2004). Low, non-amnesic doses of MDMA may also have other therapeutic effects such as increased sociality or openness that facilitate the clinical improvements observed following MDMA-assisted psychotherapy by, for example, improving the doctor-patient therapeutic alliance (Heifets and Malenka 2016; Wagner et al. 2017).

There is increasing evidence that the therapeutic effects of MDMA are mediated by the serotonergic system whereas its addiction-related effects are primarily mediated by the dopaminergic system. Young et al. (2017) demonstrated that the action of MDMA at SERT and subsequent 5-HT2A receptor activation plays an important role in its enhancement of fear memory extinction. Similarly, Heifets et al. (2019) demonstrated that the action of MDMA at SERT and subsequent 5-HT1B receptor activation within the nucleus accumbens is necessary and sufficient for its prosocial effects, whereas MDMA binding at DAT and the consequent increase in DA release is required for its rewarding effects. Risbrough et al. (2006) revealed that the DA receptor subtypes have differential modulatory roles in MDMA-induced hyperactivity; specifically, D1 receptor activation modifies the type of activity (linear versus circumscribed) whereas D2 receptor activation contributes to repetitive circling behavior. It is generally accepted that DAT is responsible for the addictive properties of MDMA, although there is some evidence that SERT may play a lesser role (Trigo et al. 2007). In any case, the evidence is that MDMA use does not often result in addiction (Degenhardt et al. 2010).

The effects of MDMA on the serotonergic versus dopaminergic systems are also dose-dependent. At low doses (< 3 mg/kg), MDMA stimulates 5-HT release and little to no DA release, whereas at high doses (≥ 3 mg/kg), MDMA stimulates both 5-HT and DA release (Baumann et al. 2005, 2007; Kankaanpää et al. 1998). In accordance with these findings, we detected MDMA-induced addiction-related behaviors at high doses that correlate with substantial DA release. Additional evidence suggests that the R(-) enantiomer of MDMA retains the prosocial and fear extinction-enhancing effects but possibly not the abuse liability of racemic MDMA because of its significantly decreased potency as a DA releaser (Pitts et al. 2018). It is plausible that low-dose racemic MDMA or another drug that preferentially induces 5-HT release may promote prosocial behavior and have a significantly reduced abuse liability.

Our findings suggest that therapeutic use of MDMA below 3 mg/kg is less likely to produce significant adverse cognitive effects. While psychostimulants have the potential for addiction and toxicity at high doses, they are effective and safe cognitive enhancers that are prescribed at low doses for extended periods of time (for review, see Wood et al. 2014). Similarly, MDMA is showing promise as a psychotherapeutic, and low doses seem to pose little risk of memory impairments, addiction, or depressed mood. It is important to note that the dose threshold for potential memory impairments and addiction (3 mg/kg MDMA) is close to the doses used in recent clinical studies (approximately 1–2 mg/kg MDMA; for review, see Feduccia et al. 2018) and this may limit therapeutic viability. Future studies should consider exploring ultra-low doses of MDMA (< 1 mg/kg), which, like psychostimulants (e.g., Wood and Anagnostaras 2009), may be even safer and as effective compared to low doses (1–2 mg/kg). In all, we believe that the potential adverse effects of MDMA must be considered within the framework of its therapeutic application, with a particular orientation to the use of low doses.

Introduction

MDMA, known for its recreational use, also shows significant promise as a psychotherapeutic agent. It affects various brain systems, primarily increasing the levels of serotonin, norepinephrine, and dopamine by reversing their transporters. MDMA is chemically related to methamphetamine, but its unique behavioral effects—such as increased empathy, trust, and social interaction—distinguish it from other stimulants and hallucinogens. These prosocial effects suggest MDMA's potential to enhance psychotherapy for conditions like social anxiety, autism spectrum disorders, or even improve social behavior independently. Recent clinical trials also indicate that MDMA-assisted psychotherapy is effective for severe post-traumatic stress disorder, potentially surpassing existing treatments.

Despite its therapeutic promise, concerns exist regarding MDMA's potential for behavioral toxicity, including memory problems, addiction, and depressed mood. A significant challenge in developing psychedelic therapies is the lack of detailed information on how different doses affect outcomes. This data is crucial for determining safe and effective dosages. For instance, certain stimulants can enhance cognition at very low doses but become addictive and impairing at high doses. Previous reviews of MDMA research found no evidence of memory impairment in animals at low doses (under 3 mg/kg), but mixed results at higher doses.

To address these gaps, this study systematically examined the dose-effect relationship of MDMA. It aimed to investigate if 3 mg/kg is indeed a threshold for MDMA-induced amnesia and to understand how MDMA doses affect memory, addiction-related behaviors, and depressive-like states. The study used a broad range of MDMA doses in mice, from very low to high, including doses comparable to those used in human clinical trials.

The research utilized several well-established animal models. Pavlovian fear conditioning was used to assess learning and memory, measuring how mice learn to associate a neutral cue with an unpleasant experience. Behavioral sensitization, conditioned place preference, and conditioned responding were employed to evaluate the addictive potential of MDMA, observing how repeated drug exposure affects activity and drug-seeking behaviors. Lastly, the Porsolt forced swim test was used to model depressive-like behavior, measuring how long mice remain immobile when placed in water. These models provide insights into the behavioral impacts of MDMA across a spectrum of effects.

Methods

Subjects

A total of 184 hybrid C57BL/6Jx129S1/SvImJ mice, split almost equally between males and females, were used. This mouse strain is commonly selected for its good behavioral characteristics and genetic consistency, which helps compare results across different experiments and laboratories. Mice were housed in groups with unlimited access to food and water and were at least 10 weeks old when testing began. All animal care and experimental procedures followed approved guidelines.

Drugs

3,4-MDMA HCl was dissolved in saline and administered by intraperitoneal injection at a volume of 10 mL/kg. The chosen MDMA doses ranged from 0 (saline control) to 0.01, 0.05, 0.1, 0.5, 1, 3, and 10 mg/kg.

Fear conditioning

Mice were tested in individual chambers designed to create two distinct environments for training and testing. Freezing behavior and locomotor activity were recorded using specialized software. Mice were randomly assigned to different MDMA dose groups. An injection of MDMA or saline was given 30 minutes before a training session, which included a single tone-shock pairing. Locomotor activity before the shock and reactivity to the shock were measured. Freezing behavior after the shock was used to assess short-term memory. Seven days after training, mice were tested for long-term context memory in the training environment, and one day later, for long-term tone memory in a different environment. All long-term memory tests occurred while the mice were off the drug.

Behavioral sensitization, conditioned place preference, and conditioned responding

Mice were tested in chambers divided into two distinct sides, with different flooring and wall patterns. Activity, including locomotion, stereotypy, and vertical movements, was tracked. Mice were assigned to groups receiving 0, 0.1, 1, or 10 mg/kg MDMA. These doses were chosen to include those that showed no effects in the fear conditioning experiment (0.1 and 1 mg/kg) and one that impaired memory (10 mg/kg). Over seven days, mice were injected with saline and placed in the "unpaired" side, then injected with MDMA and placed in the "paired" side. Behavioral sensitization was measured as the change in activity on the paired side from the first to the last training day. After training, mice were tested for conditioned place preference by allowing them free access to both sides and measuring time spent and distance traveled on each side. Finally, mice underwent challenge tests with saline and then a high dose of MDMA (10 mg/kg) on the paired side, measuring activity to evaluate conditioned and sensitized responses.

Forced swim test

The forced swim test was conducted in individual cylindrical tanks filled with water. Immobility time was measured using video tracking software. Mice were assigned to MDMA dose groups of 0, 0.1, 0.5, 1, 3, or 10 mg/kg. MDMA or saline was administered 30 minutes before the 6-minute test, and immobility during the final 4 minutes was scored to assess potential antidepressant effects.

Statistical analyses

Data were analyzed using statistical methods to identify overall differences between groups. Specific tests were used for repeated measures and for individual data points. When significant group differences were found, further comparisons were made against the saline control group. Data from male and female mice were generally combined, as sex differences did not significantly alter the main findings.

Results

Fear conditioning

The effects of various MDMA doses on fear learning and memory were examined using a Pavlovian fear conditioning paradigm. During the training session, mice given 3 mg/kg MDMA showed slightly increased baseline movement compared to controls, but there were no group differences in how mice reacted to the foot shock itself. MDMA dose-dependently affected freezing behavior, which indicates memory. During the on-drug short-term memory test, only mice given 10 mg/kg MDMA showed reduced freezing. In the off-drug long-term memory tests (both context and tone memory, conducted a week later), only mice previously given 3 or 10 mg/kg MDMA showed reduced freezing compared to controls. Low doses of MDMA (0.01 to 1 mg/kg) did not impair memory.

Behavioral sensitization, conditioned place preference, and conditioned responding

The effects of MDMA on behaviors related to addiction were investigated. No significant group differences in activity were observed on the first day of training. However, by day 7, mice receiving 10 mg/kg MDMA showed significantly increased locomotor, stereotyped, and vertical activity compared to saline controls. This pattern of increased activity in the 10 mg/kg group became apparent from day 3 to day 7 of training. Furthermore, only mice receiving 10 mg/kg MDMA showed a significant increase in activity from day 1 to day 7, indicating the development of behavioral sensitization.

When tested for conditioned place preference, there were no significant differences in the distance traveled or time spent on the drug-paired side versus the unpaired side across any of the groups, meaning no group showed a clear preference for the drug-associated environment. Following training, mice were challenged with saline and a high dose of MDMA (10 mg/kg) on the paired side. Only mice previously trained with 10 mg/kg MDMA showed increased locomotion in response to both the saline challenge (conditioned responding) and the high-dose MDMA challenge (sensitized responding).

Forced swim test

The effects of MDMA on depressive-like behavior were assessed using the forced swim test. Mice given 3 or 10 mg/kg MDMA exhibited reduced immobility compared to saline controls. Lower doses of MDMA (0.1, 0.5, and 1 mg/kg) had no significant effect on immobility.

Discussion

This study highlights the critical role of dose selection in determining the behavioral effects of MDMA. High doses (3 and 10 mg/kg) led to impairments in fear memory, some evidence of addiction potential (at 10 mg/kg), and antidepressant-like effects (at 3 and 10 mg/kg). In contrast, low doses (1 mg/kg or less) did not produce these effects. This suggests a narrow viable therapeutic window for MDMA, emphasizing the importance of careful dose consideration in clinical applications. The findings further support that 3 mg/kg MDMA appears to be a threshold for memory impairment, as seen in both short-term and long-term memory tests, and these effects were not influenced by changes in pain sensitivity or acute movement. Unlike some psychostimulants, MDMA did not enhance memory, showing a profile similar to the selective serotonin reuptake inhibitor citalopram. Further investigation into low-dose MDMA's effects on fear extinction, which is relevant for therapies, is warranted.

Regarding addiction-related behaviors, low, clinically relevant doses of MDMA (0.01 and 1 mg/kg) did not induce any signs of addiction potential. However, a high dose of 10 mg/kg MDMA did lead to behavioral sensitization (increased response to repeated drug exposure) and conditioned responding (drug-like responses to environmental cues previously paired with the drug). This high dose did not cause acute motor stimulation or a clear conditioned place preference, suggesting a potentially lower rewarding effect compared to more addictive psychostimulants. While some sex differences in acute locomotor activity were observed at the highest dose, both sexes developed sensitization similarly.

The study also addressed how MDMA modulates depressive symptoms. Acute antidepressant-like effects were observed at high, memory-impairing doses of 3 and 10 mg/kg in the forced swim test. However, these effects were not seen at lower doses (0.1, 0.5, and 1 mg/kg). The absence of acute antidepressant effects at low, clinically relevant doses suggests that if low-dose MDMA has antidepressant properties, it might require chronic administration. It is also possible that the therapeutic benefits of low-dose MDMA stem from its ability to enhance sociality or openness, which could improve the patient-therapist relationship in psychotherapy.

Evidence suggests that MDMA's therapeutic effects are often linked to the serotonergic system, while its addiction-related effects are primarily mediated by the dopaminergic system. This is consistent with findings that low doses of MDMA mainly stimulate serotonin release, while high doses stimulate both serotonin and dopamine release. The addiction-related behaviors observed in this study at high doses correlate with substantial dopamine release. Overall, research indicates that MDMA use typically does not result in addiction. This study reinforces that therapeutic use of MDMA below 3 mg/kg is less likely to cause significant adverse cognitive effects or addiction concerns. Given that current clinical doses are close to this threshold, exploring ultra-low doses (below 1 mg/kg) may offer even greater safety while maintaining efficacy, similar to how psychostimulants are effectively used at low doses. The potential adverse effects of MDMA should always be considered within the framework of its therapeutic application, with a strong emphasis on the use of low doses.

Introduction

MDMA, often used recreationally, is also being investigated for its potential as a therapeutic agent. It primarily affects the brain's serotonin system, increasing levels of serotonin, norepinephrine, and dopamine. While chemically related to methamphetamine, MDMA is distinct for its "prosocial" effects, such as increased empathy, trust, and sociality. These effects suggest MDMA could enhance psychotherapy for conditions like social anxiety, autism spectrum disorders, or post-traumatic stress disorder (PTSD). Clinical studies have shown promise for MDMA-assisted psychotherapy in treating severe PTSD, potentially surpassing current approved medications.

Despite MDMA's therapeutic promise, concerns about potential behavioral side effects, such as memory problems, addiction, and depressed mood, remain. A key obstacle to its therapeutic development has been the lack of detailed dose-response data, which is crucial for determining safe and effective doses. For instance, psychostimulants can enhance cognition at low doses but may cause addiction and cognitive impairment at high doses. Previous reviews indicated MDMA did not impair animal memory at low doses (below 3 mg/kg) but showed mixed results at higher doses. To address the need for more systematic data, the current study aimed to determine if 3 mg/kg is a threshold for MDMA-induced memory loss and to better understand the dose-response relationship of MDMA on memory, addiction-related behaviors, and depression.

Dosing between animal and human studies often requires scaling, typically by body weight. While low MDMA doses (1-2 mg/kg) lead to similar drug concentrations and brain chemical release in both humans and rodents, the timing of peak effects differs, justifying temporal, but not dose, scaling. This study systematically examined MDMA's effects at doses from 0.01 to 10 mg/kg in mice. These doses cover a wide range, from one-tenth to ten times the amounts used in recent human clinical trials (approximately 1–2 mg/kg MDMA), and were applied to tests for memory, addiction-related behaviors, and depressive-like states.

Pavlovian fear conditioning is a common method for studying how drugs affect learning and memory in rodents. In this test, an animal learns to associate a neutral cue, like a tone or a specific environment, with an unpleasant event, such as a mild footshock. After learning, the animal will show a fear response, typically by "freezing," when it encounters the cue alone. Memory for both the environment and the tone involves the amygdala, while memory for the environment also involves the hippocampus. Psychostimulants affect fear memory based on the dose: low, clinically relevant doses can improve long-term memory, but high, commonly abused doses can impair it. Similarly, citalopram, a type of antidepressant, impairs fear memory only at high doses, not low ones. Research suggests that memory improvement caused by psychostimulants depends on their effects on specific brain transporters for dopamine and norepinephrine.

Behavioral sensitization, an increased response to a drug over repeated use; conditioned place preference, a preference for a drug-associated environment; and conditioned responding, drug-like reactions to drug cues, are all measures reflecting a drug's potential for addiction. Psychostimulants affect these behaviors in a dose-dependent manner: low, memory-enhancing doses show no signs of addiction potential, while high, memory-impairing doses do. The addictive properties of high-dose psychostimulants are largely due to their significant impact on dopamine levels. However, drugs with weaker effects on dopamine transporters, such as bupropion or low-dose Adderall, are less likely to cause addiction. The forced swim test is a common model for studying depressive-like behavior in rodents and is used to screen for antidepressant medications. In this test, animals are placed in water, and the time they spend actively trying to escape versus passively immobile (showing "behavioral despair") is measured. Effective antidepressant drugs typically reduce the amount of time animals spend immobile in this test.

Most published studies on MDMA's effects in these behavioral tests have used high doses, with a notable lack of low-dose or full dose-ranging investigations. Prior research on fear conditioning and the forced swim test at high MDMA doses has yielded inconsistent results regarding memory and depressive-like behaviors. While MDMA reliably causes behavioral sensitization at a range of doses and some conditioned responding, its effect on conditioned place preference has been inconsistent across previous dose-ranging studies, often only appearing at high doses. The current study specifically addresses this gap by being the first to systematically examine the effects of very low MDMA doses (1 mg/kg or less) across these tasks, and it compares the full dose-effect relationship using consistent methods.

Methods

Subjects

A total of 184 hybrid mice, comprising both male and female subjects, were included in the study. This specific mouse strain was chosen for its consistent behavioral traits, which helps with comparing research findings across different studies. The mice were housed in groups of 2-5, with constant access to food and water, under a controlled light-dark cycle. Prior to testing, all mice were at least 10 weeks old and underwent a brief handling period. The full group of 184 mice participated in the fear conditioning experiments. Subsets of these mice were later used for tests of behavioral sensitization, conditioned place preference, and conditioned responding, and a separate subset for the forced swim test, with random assignment to these subsequent experiments. Animal care and experimental procedures followed approved guidelines.

Drugs

MDMA was prepared by dissolving it in saline solution and administered by injection into the abdomen at a volume of 10 mL/kg. The study used a range of MDMA doses: 0, 0.01, 0.05, 0.1, 0.5, 1, 3, and 10 mg/kg.

Fear Conditioning

The fear conditioning protocol involved individual chambers where mice were tested. These chambers could be modified to create two distinct environments: a training context and an alternate context for different tests. Each environment had unique visual, tactile, and scent cues. Video tracking software was used to measure freezing behavior and movement.

Mice were randomly assigned to different MDMA dose groups or a saline control group, ensuring a balance of sexes and chambers. Thirty minutes before a 10-minute training session, mice received an injection of MDMA or saline. This timing was chosen to coincide with the drug's peak effects. Training started with a baseline period, followed by a single pairing of a tone and a mild footshock. After the pairing, a short test measured immediate memory by observing freezing behavior.

Seven days later, mice were tested in the training context without any drug to assess long-term memory for the environment. The following day, mice were tested in the alternate context, also without drug, to measure long-term memory for the tone stimulus. Freezing behavior was the primary measure for memory in both long-term tests.

Behavioral Sensitization, Conditioned Place Preference, and Conditioned Responding

These tests were conducted in chambers divided into two distinct sides, one associated with the drug and one not. The sides had different flooring and wall patterns. Software tracked mouse movement to measure general activity, repetitive movements, and rearing.

Mice were divided into new dose groups (0, 0.1, 1, or 10 mg/kg MDMA), chosen to represent effects observed in the fear conditioning experiment. Before training, mice became familiar with the chambers. Training lasted 7 days, with drug administration every other day: saline in the unpaired side and MDMA in the paired side. Activity in the drug-paired side was measured, and changes in activity from day 1 to day 7 were used to assess behavioral sensitization.

One day after training, mice were tested for conditioned place preference. The divider between the two sides of the chamber was removed, allowing mice to freely explore both sides. The time spent and activity on each side were recorded to see if mice preferred the drug-paired side.

Two days after training, mice underwent two challenge tests in the drug-paired side: first with saline, then with a high dose of MDMA (10 mg/kg). Activity was measured during these challenges to evaluate conditioned responding (reaction to the drug-paired side with saline) and sensitized responding (exaggerated reaction to the high MDMA dose).

Forced Swim Test

The forced swim test was conducted using individual cylindrical tanks filled with water. Video cameras and tracking software were used to measure how long mice remained immobile.

Mice were randomly assigned to different MDMA dose groups or a saline control. Thirty minutes before the test, mice received an injection of MDMA or saline. Each mouse was then placed in the water for a 6-minute test. The amount of time spent immobile during the last 4 minutes was recorded to assess potential antidepressant effects, which would be indicated by reduced immobility.

Statistical Analyses

Data were analyzed using standard statistical methods, specifically analyses of variance (ANOVAs) for comparing differences between groups, and multivariate analyses of variance (MANOVAs) for data collected repeatedly or within the same subjects. When significant group differences were found, further tests were conducted to identify which specific groups differed from the saline control. Generally, data from male and female mice were combined for analysis because no significant sex differences were observed that would alter the main findings.

Results

Fear Conditioning

The study examined how different doses of MDMA (0–10 mg/kg) affected fear learning and memory. Mice were trained after receiving the drug, and their freezing behavior was measured during an immediate post-shock test and again one week later (without drug) for long-term memory of the environment and tone.

MDMA had a minor dose-dependent effect on movement during the initial training period; only mice given 3 mg/kg MDMA showed increased movement compared to the control group. The footshock itself caused a strong response in all mice, but the intensity of this response did not differ across groups.

MDMA significantly affected freezing behavior in a dose-dependent manner during all memory tests. Compared to controls, only mice given 10 mg/kg MDMA showed reduced freezing in the immediate post-shock test, indicating impaired short-term memory. For long-term memory, only mice that had previously received 3 or 10 mg/kg MDMA showed reduced freezing in both the environment and tone tests, indicating impaired long-term memory. Lower doses of MDMA (0.01 to 1 mg/kg) did not impair memory.

Behavioral Sensitization, Conditioned Place Preference, and Conditioned Responding

The study investigated the effects of MDMA (0–10 mg/kg) on behaviors related to addiction. Mice were trained over 7 days, receiving saline in one chamber side and MDMA in another. Activity was measured in the drug-paired side. On day 1, no significant differences in activity were seen across groups. However, by day 7, mice given 10 mg/kg MDMA showed significantly increased movement, repetitive behaviors, and rearing compared to control mice, while lower doses did not.

Throughout the 7-day training period, only mice receiving 10 mg/kg MDMA showed a progressive increase in activity (movement, repetitive behaviors, and rearing), particularly from day 3 to day 7. This indicated the development of behavioral sensitization, meaning their response to the drug became stronger over time.

Following training, mice were tested for conditioned place preference without drug. They could freely access both sides of the chamber. No group showed a significant preference for the drug-paired side in terms of distance traveled or time spent. This suggests that MDMA, at the tested doses, did not strongly create a rewarding association with the environment.

In follow-up challenge tests, mice that had been trained with 10 mg/kg MDMA showed increased movement when given a saline injection in the drug-paired environment, indicating a conditioned response. These mice also showed a strong sensitized response, meaning a much higher level of movement, when subsequently given a high dose of MDMA (10 mg/kg). Similar patterns were observed for repetitive behaviors, but not for rearing.

Forced Swim Test

MDMA affected immobility in the forced swim test in a dose-dependent manner. Mice given 3 or 10 mg/kg MDMA exhibited significantly reduced immobility compared to the saline control group, suggesting a potential antidepressant effect at these higher doses. Lower MDMA doses (0.1, 0.5, 1 mg/kg) did not show this effect.

Discussion

This study reinforces the critical importance of dose selection when considering the behavioral effects of MDMA. High doses of MDMA (3 and 10 mg/kg) were found to impair fear memory, show some indication of addiction potential (at 10 mg/kg), and produce antidepressant-like effects (at 3 and 10 mg/kg). In contrast, low doses of MDMA (1 mg/kg or less) did not produce these effects. This suggests that frequent high-dose MDMA should probably be avoided due to its potential for memory problems and addiction. However, low-dose MDMA (around 1–2 mg/kg), similar to doses used in recent clinical trials, appears to be safe concerning the behaviors examined in this study. Overall, MDMA seems to have a narrow therapeutic range, highlighting the need to carefully consider dose reduction in clinical applications.

Previous research had questioned whether MDMA's therapeutic use could cause memory problems, especially at low doses. This study provides stronger evidence that 3 mg/kg MDMA appears to be the threshold for memory impairment. High doses (3 and 10 mg/kg) impaired both short-term and long-term fear memory. These memory deficits were not due to changes in pain sensitivity or general movement, suggesting they reflect actual memory impairment, possibly an inability to form new memories. Unlike some psychostimulants, MDMA did not enhance fear memory at any dose; its memory effects were more similar to antidepressants like citalopram, which only cause impairment at high doses. While MDMA-assisted psychotherapy for PTSD might involve enhancing fear extinction, this study did not observe changes in fear learning at low, clinically relevant doses, though higher doses have been reported to improve fear memory extinction, warranting further research.

The study found that low, clinically relevant doses of MDMA (0.01 and 1 mg/kg) did not lead to any behaviors associated with addiction, even when challenged with a high MDMA dose. However, a high, memory-impairing dose of 10 mg/kg MDMA did lead to behavioral sensitization (an increased response with repeated use) and conditioned responding (reaction to drug-associated cues), but not an immediate increase in general movement or conditioned place preference (preference for the drug environment). While previous studies have shown varying results for MDMA-induced place preference, the weak effect observed here, even if statistically significant, was much smaller compared to what is typically seen with psychostimulants. This suggests that while repeated high-dose MDMA use might lead to compulsive drug-taking and craving, MDMA may be less rewarding and less likely to cause drug-seeking behavior than more potent psychostimulants. It appears that even at high doses, MDMA is only a mild stimulant for general movement. Some sex differences were observed, with females showing increased movement earlier in training at the 10 mg/kg dose, although both sexes developed sensitization similarly.

Regarding depressive-like behavior, the study found acute antidepressant-like effects with high MDMA doses (3 and 10 mg/kg), indicated by reduced immobility in the forced swim test. No such effects were seen at lower doses (0.1, 0.5, and 1 mg/kg). It is important to note that drugs which acutely increase movement can sometimes lead to false positive results in the forced swim test. However, given that high doses of MDMA only mildly stimulated movement in this study, it is unlikely that this was the sole cause of the reduced immobility. Since low, clinically relevant doses did not show acute antidepressant effects, it is possible that lower doses of MDMA might require repeated administration to reduce depressive-like behavior, similar to how conventional antidepressants work. Additionally, low, non-memory impairing doses of MDMA might have other therapeutic benefits, such as increasing sociality or openness, which could improve the patient-therapist relationship in MDMA-assisted psychotherapy.

Evidence indicates that MDMA's therapeutic effects are primarily mediated by the serotonin system, while its addiction-related effects are linked to the dopamine system. The impact of MDMA on these systems is dose-dependent: low doses (below 3 mg/kg) mainly increase serotonin release, while high doses (3 mg/kg or more) significantly increase both serotonin and dopamine. The addiction-related behaviors observed at high doses in this study support this link to substantial dopamine release. These findings suggest that MDMA's therapeutic use below 3 mg/kg is less likely to produce significant adverse cognitive effects. While the threshold for potential memory impairments and addiction (3 mg/kg) is close to current clinical doses (1–2 mg/kg), indicating a narrow therapeutic window, exploring ultra-low doses (below 1 mg/kg) could offer an even safer and effective approach. In conclusion, MDMA's potential adverse effects should always be considered within its therapeutic context, with a clear emphasis on the careful application of low doses.

Introduction

MDMA, known recreationally as ecstasy, is also being studied for its potential as a treatment in psychotherapy. It affects various brain chemicals and pathways, especially those involving serotonin. MDMA increases levels of serotonin, norepinephrine, and dopamine by changing how their transporters work. While chemically similar to methamphetamine, MDMA is often described as an "empathogen" or "entactogen" due to its ability to increase empathy, trust, social interaction, and openness. These social effects are what make MDMA particularly interesting for treating conditions like social anxiety and autism spectrum disorders, and potentially as a stand-alone treatment to improve social behavior. Recent clinical trials suggest that MDMA-assisted therapy is effective for severe post-traumatic stress disorder (PTSD), potentially performing better than current standard treatments.

Despite its therapeutic promise, there are some worries about MDMA's effects on behavior, such as memory problems, addiction, and low mood. A significant challenge in developing psychedelic therapies is the lack of clear information on how different doses affect people. This information is crucial for determining safe and effective doses. For example, stimulant medications can improve thinking at very low doses but become addictive and harmful to thinking at high doses. Previous research found no evidence that MDMA harms memory at low doses (less than 3 mg/kg) in animals, but results for high doses were mixed. This study aims to further understand how different doses of MDMA affect memory, addiction, and depression, particularly exploring if 3 mg/kg is indeed the point at which memory issues begin.

The doses of MDMA used in animals are generally scaled based on body weight to compare them to human doses. Low doses of MDMA (about 1 to 2 mg/kg) create similar drug levels and chemical release in the brain in both humans and rodents. However, the drug reaches its peak effect faster in rodents. This study systematically looks at the effects of a wide range of MDMA doses (0.01 to 10 mg/kg) on mice, covering doses both lower and higher than those used in current human clinical trials (which are around 1–2 mg/kg). The study examines MDMA's effects on fear conditioning (a measure of learning and memory), as well as behaviors related to addiction, and a test for depressive-like behavior.

Methods

Subjects

A total of 184 male and female mice were used. These mice were chosen because they are a standard strain that helps researchers compare results across different studies and labs, and they tend to have good behavioral traits. The mice were housed in groups with unlimited food and water. They were kept on a regular light and dark cycle, and all tests happened during the light period. Mice were at least 10 weeks old and were handled for a few days prior to testing. All mice were initially used for memory tests. Weeks later, some of these mice were randomly selected for further tests related to addiction and depressive-like behavior. All animal care and study procedures followed strict guidelines and were approved by the university's animal care committee.

Drugs

MDMA was prepared by dissolving it in a saline solution and given to mice by injection into the abdominal cavity. A range of MDMA doses was tested: 0 (saline only), 0.01, 0.05, 0.1, 0.5, 1, 3, and 10 mg/kg.

Fear conditioning

The study used a system that tracks mouse movement to measure fear conditioning. Four mice were tested at a time in separate chambers. These chambers were designed to create two distinct environments: one for training and memory testing, and another for a different type of memory test. The environments were made different by changing the lighting, scents, and floor types. Tracking software recorded how much the mice "froze" (a common fear response) and how much they moved.

Mice were randomly assigned to different MDMA dose groups. They received an injection of MDMA or saline 30 minutes before a 10-minute training session. This 30-minute delay matched the time when the drug typically reaches its peak effect in mice. The training involved a three-minute baseline, followed by a specific tone that ended with a brief electric foot shock. After this, mice had a short test period to measure immediate memory while still under the drug's effect. Seven days later, the mice were tested again in the training environment, without the drug, to measure long-term memory related to the environment. The next day, they were tested in the alternate environment, also without the drug, to measure long-term memory related to the tone.

Behavioral sensitization, conditioned place preference, and conditioned responding

These tests were done using special chambers with two distinct sides, separated by a removable wall. The two sides looked and felt different, with various flooring and wall designs. Tracking software recorded mouse position and movement. Forty-five mice were randomly assigned to new groups for these tests, receiving doses of 0, 0.1, 1, or 10 mg/kg MDMA. These doses were chosen to include ones that had no effect on memory, and one that did cause memory problems in the previous fear conditioning experiment.

Mice were accustomed to the testing chamber for two days before training began. Training lasted seven days, with sessions every other day. On each training day, mice received a saline injection before being placed in one side of the chamber, then an MDMA injection before being placed in the other side. Researchers measured their movement, repetitive behaviors, and standing on hind legs in the MDMA-paired side. "Behavioral sensitization" was measured by comparing activity on the first day to activity on the last day, indicating a progressive increase in response to the drug.

Twenty-four hours after the final training day, mice were tested for "conditioned place preference" without any drug. The dividing wall in the chamber was removed, allowing mice to move freely between the two sides for 15 minutes. Researchers measured the time spent and distance traveled on each side to see if mice preferred the side where they had received MDMA, which indicates the drug's rewarding effects.

Forty-eight hours after the last training day, mice had two challenge tests. First, they received a saline injection and were placed in the MDMA-paired side. Then, they received a high dose of MDMA (10 mg/kg) and were returned to the MDMA-paired side. Movement and other behaviors were scored during these challenges to evaluate "conditioned responding" (a drug-like response to the drug-paired environment without the drug) and "sensitized responding" (an exaggerated response to a high dose of the drug after repeated exposure).

Forced swim test

This test is used to identify behaviors similar to depression and to screen for antidepressant drugs in rodents. Mice were placed in individual glass tanks filled with water. Their activity was recorded using a video camera and tracking software. Researchers measured how much time the mice spent immobile (a sign of "behavioral despair" or giving up) versus active (trying to escape).

Seventy-nine mice were randomly assigned to new groups and received doses of 0, 0.1, 0.5, 1, 3, or 10 mg/kg MDMA. They were given the injection 30 minutes before the test. Mice were placed in the water for six minutes, and the time they spent immobile during the last four minutes was measured to see if MDMA had antidepressant-like effects (reduced immobility).

Statistical analyses

The data were analyzed using standard statistical methods to identify overall differences between groups. These methods helped determine if the observed differences were likely due to the MDMA treatment or just random chance. If significant differences were found, further tests were conducted to pinpoint which specific groups differed from the control group (saline). In most cases, data from male and female mice were combined because there were no significant sex differences that changed the main findings.

Results

Fear conditioning

This study looked at how MDMA (doses from 0 to 10 mg/kg) affected learning and memory related to fear. Mice were trained while under the influence of the drug. Freezing behavior was measured immediately after the training (short-term memory) and then one week later (long-term memory in the environment) and one day after that (long-term memory related to the tone), all while off the drug.

MDMA had a small effect on how much mice moved before the training started. Only mice given 3 mg/kg MDMA moved more than those given saline. The foot shock caused a strong reaction in all mice, and this reaction was similar across all groups, meaning MDMA didn't change how mice reacted to the shock itself. However, MDMA significantly affected freezing behavior in all memory tests. Only mice given 10 mg/kg MDMA showed reduced freezing during the immediate post-shock test. For long-term memory, mice that had previously received 3 or 10 mg/kg MDMA showed reduced freezing in both the environment and tone tests compared to saline-treated mice. This suggests that these higher doses impaired long-term fear memory. Lower doses (0.01 to 1 mg/kg) did not impair memory.

Behavioral sensitization, conditioned place preference, and conditioned responding

The study also investigated how MDMA (doses 0 to 10 mg/kg) affected behaviors linked to addiction. Mice were trained over seven days, receiving saline in one chamber side and MDMA in another. Their activity in the MDMA-paired side was measured. On the first day of training, there were no significant differences in activity levels among the groups. However, by day seven, only mice receiving 10 mg/kg MDMA showed significantly increased movement, repetitive behaviors, and vertical activity compared to saline-treated mice.

Over the seven days of training, only the 10 mg/kg MDMA group showed increasing activity levels from day three to day seven, indicating the development of "behavioral sensitization," meaning their reaction to the drug became stronger over time. This group also showed a greater increase in activity from day one to day seven compared to the saline group.

After training, mice were tested for "conditioned place preference," where they could freely move between the two chamber sides. There were no significant differences in how much distance mice traveled or time they spent on either the drug-paired or unpaired sides across any of the groups. None of the groups showed a strong preference for the side where they had received MDMA.

Finally, mice were given challenge injections in the drug-paired side. When given a saline challenge, only mice previously trained with 10 mg/kg MDMA showed increased movement, suggesting "conditioned responding" (a learned response to the environment associated with the drug). When given a high dose of MDMA (10 mg/kg) challenge, only the mice previously trained with 10 mg/kg MDMA showed increased movement, indicating "sensitized responding" (an exaggerated reaction to the high dose due to prior exposure). Similar patterns were seen for repetitive behaviors but not for vertical activity.

Forced swim test

The effects of MDMA (doses 0 to 10 mg/kg) on depressive-like behavior were assessed using the forced swim test. Mice were placed in water, and the time they spent immobile was measured. MDMA significantly affected immobility depending on the dose. Only mice given 3 or 10 mg/kg MDMA showed reduced immobility compared to saline-treated mice, suggesting these higher doses had an antidepressant-like effect.

Discussion

This study highlights that the effects of MDMA on behavior are strongly tied to the dose given. High doses (3 and 10 mg/kg) led to problems with fear memory, some signs of addiction (at 10 mg/kg), and antidepressant-like effects. In contrast, low doses of MDMA (1 mg/kg or less) did not cause these behavioral changes. This suggests that MDMA has a limited dose range where it might be therapeutically useful, and careful attention to dose is crucial for its clinical application.

The findings support earlier suggestions that 3 mg/kg MDMA might be the point at which memory issues begin. This study specifically showed that 10 mg/kg MDMA impaired short-term memory, and both 3 and 10 mg/kg MDMA impaired long-term memory related to context and tone. Importantly, these memory problems were not due to changes in how mice reacted to pain or how much they moved before training. Lower doses (0.01 to 1 mg/kg) did not harm memory. Unlike some stimulant drugs, MDMA did not enhance memory at low doses, behaving more like certain antidepressant medications that impair memory only at high doses. Further research is needed to explore if low-dose MDMA could help improve fear extinction, a process relevant to PTSD treatment.

Regarding addiction-related behaviors, low, clinically relevant doses of MDMA (0.01 and 1 mg/kg) did not lead to any signs of addiction, even when challenged with a high MDMA dose. However, a high, memory-impairing dose of 10 mg/kg MDMA did cause behavioral sensitization (increased response after repeated use) and conditioned responding (learned response to a drug-related environment). This dose did not cause an immediate increase in movement or "conditioned place preference" (a preference for the environment where the drug was given). While some previous studies reported place preference with similar high doses, the effect observed here was much weaker compared to other stimulant drugs, suggesting MDMA might be less rewarding and less likely to cause drug-seeking behavior than typical stimulants. Interestingly, female mice showed increased activity at 10 mg/kg earlier than males, although both sexes eventually developed sensitization.

In the forced swim test, which models depressive-like behavior, high doses of MDMA (3 and 10 mg/kg) reduced immobility, suggesting antidepressant-like effects. This effect was not observed at lower doses. While some drugs that increase movement can skew forced swim test results, it is unlikely that this was the case here, as a single dose of 3 or 10 mg/kg MDMA did not significantly increase general movement. The therapeutic effects of MDMA are thought to involve the serotonin system, while its potential for addiction is linked to the dopamine system. At low doses, MDMA mainly increases serotonin release, but at high doses, it increases both serotonin and dopamine. These findings align with the idea that MDMA's addictive potential appears only at doses that cause significant dopamine release.

Overall, the findings suggest that using MDMA therapeutically at doses below 3 mg/kg is less likely to cause significant negative effects on memory. Similar to how stimulants are safely prescribed at low doses for long periods, MDMA shows promise as a therapy, with low doses appearing to pose little risk of memory problems, addiction, or depressed mood. The threshold for potential negative effects (3 mg/kg) is close to the doses used in current human studies (around 1–2 mg/kg), which might limit its therapeutic use. Future studies should consider exploring even lower doses (less than 1 mg/kg), as these might be safer and just as effective as the current low doses being studied. It is important to consider the possible negative effects of MDMA within the context of its therapeutic benefits, focusing particularly on the use of low doses.

Introduction

MDMA is a drug that many people use for fun. But it is also being studied to see if it can help people in therapy, especially for problems like strong fear after bad events (PTSD) or social shyness. MDMA works by changing brain chemicals, especially one called serotonin. These changes can make people feel more open, trusting, and connected to others. This is why doctors are interested in using it to help people heal.

However, there are worries about MDMA. Some concerns include whether it can cause memory problems, lead to addiction, or make people feel sad. Doctors need to find the right dose of MDMA that is safe and helpful. Because past studies have looked at different doses in various ways, more clear research is needed to understand how different amounts of MDMA affect the body and mind.

This study looked at how different amounts of MDMA affect memory, addiction, and signs of sadness in mice. The goal was to understand which doses might be helpful and which might cause problems, especially since some doses used in this study are similar to those used in human medical trials.

Methods

Subjects

A total of 184 mice were used for this study. They were a mix of male and female mice chosen because they are good for research and help compare results with other studies. The mice lived in groups and had plenty of food and water. They were at least 10 weeks old before the study began. All mice were used for memory tests. Some of these mice were later used for tests about addiction, and others for tests about sadness. The care for all animals and the study steps were approved by a special committee.

Drugs

MDMA was mixed in salt water and given to the mice through a shot in their belly. Different amounts of MDMA were used: 0, 0.01, 0.05, 0.1, 0.5, 1, 3, and 10 mg per kilogram of body weight.

Fear conditioning

A special system and method were used to test how MDMA affects learning and memory in mice. Mice were tested one at a time in clear boxes that could be changed to look like two different places. One place was used for training, and the other for later tests.

The mice were given MDMA or plain salt water 30 minutes before training. During training, a sound was played, and then a small foot shock was given. This teaches the mice to be afraid of the sound and the place. We measured how much the mice moved before the shock and how much they froze afterward. Freezing is a sign of fear memory. One week later, the mice were put back in the training place to test their long-term memory of the place. The next day, they were put in the second place to test their long-term memory of the sound.

Behavioral sensitization, conditioned place preference, and conditioned responding

These tests looked at how likely MDMA was to cause addiction. Mice were tested in chambers with two different sides. One side was connected to the drug (paired side), and the other was not (unpaired side).

Mice were given MDMA every other day for 7 days. On training days, they got plain salt water and were put in the unpaired side, then got MDMA and were put in the paired side. We watched how much they moved, how much they did repeated actions, and how much they stood up on the drug-paired side. This helped us see if they became more sensitive to the drug over time.

One day after the last training, mice were allowed to move freely between both sides of the chamber without any drug. We checked which side they preferred to spend time on. Two days after training, mice were given challenges. They were given plain salt water on the paired side, and then a high dose of MDMA on the paired side. This showed if they had learned to react to the drug-paired place or if they were more sensitive to the drug.

Forced swim test

This test checked for signs of sadness in mice. Mice were placed in tall glass tanks filled with water. They were given MDMA or plain salt water 30 minutes before the test. We measured how long the mice stayed still (immobile) in the water. Being still for a long time can be a sign of sadness or giving up.

Statistical analyses

The information gathered was carefully reviewed to find differences between the groups. Results from male and female mice were generally combined because there were no important differences between them.

Results

Fear conditioning

The study looked at how different amounts of MDMA (0 to 10 mg/kg) affected how mice learned and remembered fear. Mice were trained while on the drug with a sound and a shock. Their freezing behavior was checked right after the shock to see short-term memory, and then again one week later in the training place and the next day with the sound, to check long-term memory. Mice given 3 mg/kg MDMA moved more than others before the shock. The shock itself caused a burst of activity, but this was the same for all groups. For short-term memory, only mice given 10 mg/kg MDMA showed worse memory. For long-term memory, mice given 3 or 10 mg/kg MDMA showed worse memory of both the place and the sound. Lower doses of MDMA (0.01 to 1 mg/kg) did not cause memory problems.

Behavioral sensitization, conditioned place preference, and conditioned responding

The study also looked at how different amounts of MDMA (0 to 10 mg/kg) affected behaviors related to addiction. Mice were trained for 7 days in a special chamber, getting MDMA on one side and plain salt water on the other. On the first day, there were no big differences in how much the mice moved or did repeated actions. But on day 7, only mice given 10 mg/kg MDMA moved more and showed more repeated actions. This increase in activity over days showed that these mice were becoming more sensitive to the drug.

After training, mice were tested for a drug-related preference for one side of the chamber. No group showed a strong preference for either side. Later, mice were given a challenge with plain salt water or a high dose of MDMA on the drug-paired side. Only mice that had been given 10 mg/kg MDMA during training moved more when given plain salt water (showing they reacted to the drug-paired place) and also moved more when given the high dose of MDMA (showing they were more sensitive to the drug).

Forced swim test

The study checked how different amounts of MDMA (0 to 10 mg/kg) affected behaviors related to sadness. Mice were placed in water, and the time they spent staying still was measured. Mice given 3 or 10 mg/kg MDMA spent less time staying still than other mice, which means these doses reduced signs of sadness. Lower doses did not have this effect.

Discussion

This study clearly shows that the amount of MDMA given is very important for its effects on behavior. High doses (3 and 10 mg/kg) caused problems with memory and some signs of addiction. However, these high doses also reduced signs of sadness. Low doses of MDMA (1 mg/kg or less) did not cause these unwanted effects. This means that when using MDMA for therapy, choosing the right, low dose is very important to keep people safe.

Previous research suggested that MDMA might not cause memory problems at low doses, but higher doses might. Our study supports this idea, showing that 3 mg/kg MDMA appears to be the amount where memory issues begin. Doses of 10 mg/kg MDMA caused problems with both short-term and long-term memory, while low doses did not. This study did not find that MDMA improved memory. For addiction-like behaviors, low doses of MDMA (0.01 and 1 mg/kg) did not lead to any signs of addiction, even when mice were later given a high dose. However, mice given a high dose of 10 mg/kg MDMA did show signs of becoming more sensitive to the drug and reacting to the drug-paired environment.

In the test for sadness, high doses of MDMA (3 and 10 mg/kg) reduced signs of sadness in mice. But low doses (0.1, 0.5, and 1 mg/kg) did not have this effect when given only once. It's possible that low doses of MDMA might need to be given over a longer time to help with sadness, similar to other antidepressant medicines. It is also believed that low-dose MDMA can help people in therapy by making them more social and open, which can improve their healing.

MDMA affects different brain chemicals depending on the dose. At low doses (less than 3 mg/kg), MDMA mainly boosts serotonin, which is linked to good feelings and social behavior. At high doses (3 mg/kg or more), it boosts both serotonin and dopamine. Dopamine is often linked to addiction. This explains why low doses are less likely to cause addiction. Overall, MDMA does not seem to lead to addiction very often.

Our findings suggest that MDMA can be used in therapy with fewer risks if low doses (under 3 mg/kg) are used, as these doses are less likely to cause memory problems or addiction. The doses used in recent human studies are close to this risk level (around 1-2 mg/kg). Therefore, it is important for future research to explore even lower doses of MDMA (less than 1 mg/kg). Like other helpful drugs that can be risky at high doses but safe at low doses, MDMA's possible negative effects should always be weighed against its benefits, with a focus on using the lowest effective doses.