Inhibition of serotonin transporters disrupts the enhancement of fear memory extinction by 3,4-methylenedioxymethamphetamine (MDMA)

This vehicle-controlled animal study (n=360) investigated the role of serotonergic neurotransmission in MDMA’s (7.8 mg/kg) ability to extinguish fearful memories in mice and found that the selective inhibition of serotonin release via 5-HTT and the inhibition of neurotransmission via 5-HT_2A receptors diminished this effect.

Abstract

“Rationale: 3,4-Methylenedioxymethamphetamine (MDMA) persistently improves symptoms of post-traumatic stress disorder (PTSD) when combined with psychotherapy. Studies in rodents suggest that these effects can be attributed to enhancement of fear memory extinction. Therefore, MDMA may improve the effects of exposure-based therapy for PTSD, particularly in treatment-resistant patients. However, given MDMA’s broad pharmacological profile, further investigation is warranted before moving to a complex clinical population.

Objectives: We aimed to inform clinical research by providing a translational model of MDMA’s effect, and elucidating monoaminergic mechanisms through which MDMA enhances fear extinction.

Methods: We explored the importance of monoamine transporters targeted by MDMA to fear memory extinction, as measured by reductions in conditioned freezing and fear-potentiated startle (FPS) in mice. Mice were treated with selective inhibitors of individual monoamine transporters prior to combined MDMA treatment and fear extinction training.

Results: MDMA enhanced the lasting extinction of FPS. Acute and chronic treatment with a 5-HT transporter (5-HTT) inhibitor blocked MDMA’s effect on fear memory extinction. Acute inhibition of dopamine (DA) and norepinephrine (NE) transporters had no effect. 5-HT release alone did not enhance extinction. Blockade of MDMA’s effect by 5-HTT inhibition also downregulated 5-HT_2A-mediated behavior, and 5-HT_2A antagonism disrupted MDMA’s effect on extinction.

Conclusions: We validate enhancement of fear memory extinction by MDMA in a translational behavioral model, and reveal the importance of 5-HTT and 5-HT_2A receptors to this effect. These observations support future clinical research of MDMA as an adjunct to exposure therapy, and provide important pharmacological considerations for clinical use in a population frequently treated with 5-HTT inhibitors.”

Authors: Matthew B. Young, Seth D. Norrholm, Lara M. Khoury, Tanja Jovanovic, Sheila AM Rauch, Collin M. Reiff, Boadie W. Dunlop, Barbara O. Rothbaum & Leonard L. Howe

Summary

Abstract

3,4-Methylenedioxymethamphetamine (MDMA) improves symptoms of post-traumatic stress disorder (PTSD) when combined with psychotherapy. This study aimed to provide a translational model of MDMA’s effect and elucidate monoaminergic mechanisms through which MDMA enhances fear extinction.

MDMA enhanced the lasting extinction of fear-potentiated startle (FPS) in mice. Acute and chronic treatment with a 5-HT transporter (5-HTT) inhibitor blocked MDMA’s effect on fear memory extinction.

MDMA enhances fear memory extinction in a translational behavioral model. 5-HTT and 5-HT2A receptors are important for this effect.

Introduction

MDMA has been used as an adjunct to psychotherapy for several decades. It has been found to promote long-term reductions in symptoms of post-traumatic stress disorder.

Many PTSD patients continue to experience intensely fearful responses to traumatic memories long after the fear response serves a protective function. MDMA may enhance the extinction of fear memory in rodents through signaling through brain-derived neurotrophic factor.

MDMA targets monoaminergic transporters and receptors in the brain, and increases 5-HT and NE neurotransmission. SSRIs and NRIs inhibit MDMA’s behavioral/subjective effects, demonstrating that MDMA’s unique behavioral profile depends on access to transporters of 5-HT and NE.

MDMA enhances extinction of fear memory through monoaminergic mechanisms. Fear-potentiated startle (FPS) is a highly conserved fear behavior across species, and its expression depends on the amygdala, where MDMA increases BDNF signaling important for learning.

We used auditory Pavlovian fear conditioning to instate a fear memory and demonstrated that MDMA enhanced the extinction of this memory. We also revealed that 5-HT transporters were crucial for this effect.

Animals

Male C57BL/6 mice were bred at the Yerkes National Primate Research Center at Emory University and were trained and tested during the lights-on phase, when rates of fear extinction are comparatively slower.

Tocris Bioscience, Sigma-Aldrich, MDMA, citalopram, reboxetine, and DOI were dissolved in 0.9% saline and injected intraperitoneally (i.p.) at a volume of 10 L/g body weight. Drug vehicle served as control for each respective experiment with a given drug.

Conditioning and extinction of freezing behavior

Mice were exposed to cued fear conditioning on day 1, fear extinction training on day 3, and extinction testing on day 4. The effect of chronically administered citalopram on freezing was examined.

Fear-potentiated startle

Studies exploring FPS were carried out using a startle response system from San Diego Instruments. MDMA doses were tested on the within- and between-session extinction of FPS.

On day 4, mice were given a 5-min acclimation period followed by 10 startle stimuli (85 dB; 1 min ITI), and then 20 BCS + startle trials. On day 5, animals were fear conditioned to 10 CS-US pairings (0.25 s, 0.4-mA) in the same fear conditioning chambers used in previously described experiments.

In the second experiment, chronic citalopram treatment was tested on the effects of MDMA. Startle was assessed in 100-ms blocks during the presentation of the startle stimulus, and acclimation periods were assessed every 30 s.

Statistical analysis

When administered alone 60 min prior to fear extinction training, citalopram, reboxetine, or RTI-336 had no effect on conditioned freezing or fear extinction.

When mice were pre-treated with citalopram or reboxetine prior to MDMA, the reduction in conditioned freezing observed in the vehicle-treated group was prevented. However, only mice treated with citalopram prior to MDMA exhibited significantly greater conditioned freezing than mice that received vehicle prior to MDMA.

MDMA’s effects on fear extinction are blocked by chronic daily injections of citalopram

Chronic administration of citalopram abolished the effect of MDMA on fear extinction. The effect was observed in both extinction training and testing, with citalopram treatment abolishing the effect of MDMA on average conditioned freezing across the whole extinction testing session.

A separate group of fear-conditioned mice was treated with vehicle or citalopram 24 h prior to extinction training and MDMA treatment. Citalopram did not affect the persistent reductions in conditioned freezing observed in MDMA-treated mice during extinction testing.

Transporter-mediated 5-HT release is insufficient to enhance extinction: a role for the 5-HT2A receptor

Fenfluramine, a compound that selectively releases 5-HT through the 5-HTT, increased conditioned freezing during extinction training. However, the effects were not maintained the following day.

Mice treated chronically with citalopram exhibited less DOI-induced head-twitch behavior and less conditioned freezing behavior 24 h after the last dose of chronic treatment. The selective 5-HT2AR antagonist M100 attenuated these effects.

Chronically administered citalopram blocks MDMA’s enhancement of the extinction of fear-potentiated startle

All three doses of MDMA administered prior to extinction training reduced FPS during the training session, but only the 7.8 mg/kg dose of MDMA significantly reduced FPS during retention of extinction the following day.

Chronic daily injections of citalopram (10 mg/kg; i.p.) for 22 days did not alter fear perception in mice, but acute MDMA treatment significantly reduced fear perception in mice, regardless of whether they had been chronically treated with either citalopram or vehicle.

Discussion

MDMA enhances the extinction of powerful fear memories, and this effect can be impaired by pharmacologically inhibiting 5-HTT.

MDMA enhances the extinction of both conditioned freezing and the acoustic startle reflex in the amygdala, and FPS appears to extinguish more slowly than conditioned freezing. This is important because FPS is frequently used as an index of conditioned fear expression and extinction in human investigations.

Although administering MDMA prior to extinction training reduces conditioned fear expression, these reductions do not predict lasting improvements in extinction learning. This suggests that subjective or behavioral responses to MDMA during extinction training may not predict lasting fear extinction outcomes.

MDMA increases levels of 5-HT in the brain, but this increase is unlikely to account for MDMA’s effect on fear memory extinction. Additionally, citalopram, a drug that increases 5-HT, does not enhance fear memory extinction.

MDMA affects several neuromodulatory systems and targets in the brain that have been previously associated with fear memory extinction. However, reboxetine did not affect MDMA’s effect on extinction in the current study, nor did it affect extinction learning on its own.

Although MDMA releases significant levels of dopamine, other transporter-mediated dopamine releasers such as amphetamine have not been demonstrated to promote extinction learning here or elsewhere. However, indirect dopamine release via the D2 receptor may play an important role in MDMA’s effect on extinction.

Chronic SSRI treatment likely inhibits MDMA-induced fear memory extinction because citalopram has an estimated half-life of 90 min in mice. Chronic SSRI treatment downregulates 5-HTT availability and function, which could diminish MDMA’s effect on fear memory extinction. However, we also explored mechanisms besides 5-HT release, including 5-HT2A receptor function, which was significantly reduced 48 h after chronic citalopram treatment.

We observed no lasting impairments in fear memory extinction in response to 5-HT2A receptor antagonist treatment alone. However, fear memory extinction is consistently enhanced by 5-HT2A receptor agonists. MDMA increases brain-derived neurotrophic factor and reduces fear expression in the amygdala, and also binds with weaker affinity at 5-HT1A and 5-HT2C receptor sites. However, 5-HT1A antagonism does not inhibit MDMA’s effect in the amygdala or on the cognitive and subjective effects of MDMA in humans.

Chronic SSRI treatment blocks MDMA’s effect on fear memory extinction by downregulating 5-HTTs and 5-HT2A receptor signaling. However, acutely administered citalopram also blocked MDMA’s effect, which may be due to rapid de-sensitization of the 5-HT2A receptor response to MDMA following citalopram treatment.

MDMA has been observed to improve psychotherapeutic outcomes in PTSD patients, possibly by enhancing the extinction of the fear response to memories for trauma. However, a portion of patients remain unresponsive to exposure-based therapies.