3,4-Methylenedioxymethamphetamine facilitates fear extinction learning

This rodent study appraises the psychotherapeutic gains facilitated by MDMA and investigates its effects on fear extinction learning, which is a key process in exposure-based therapies for PTSD. The authors propose that MDMA improves fear memory extinction via a BDNF-dependent mechanism. The study highlighted the potential of MDMA as a useful adjunct to exposure-based therapies for PTSD and other anxiety disorders marked by altered fear learning.

Abstract

“Acutely administered 3,4-methylenedioxymethamphetamine (MDMA, ‘ecstasy’) has been proposed to have long-term positive effects on post-traumatic stress disorder (PTSD) symptoms when combined with psychotherapy. No preclinical data support a mechanistic basis for these claims. Given the persistent nature of psychotherapeutic gains facilitated by MDMA, we hypothesized that MDMA improves fear extinction learning, a key process in exposure-based therapies for PTSD. In these experiments, mice were first exposed to cued fear conditioning and treated with drug vehicle or MDMA before extinction training 2 days later. MDMA was administered systemically and also directly targeted to brain structures known to contribute to extinction. In addition to behavioral measures of extinction, changes in mRNA levels of brain-derived neurotrophic factor (Bdnf) and Fos were measured after MDMA treatment and extinction. MDMA (7.8 mg kg⁻¹) persistently and robustly enhanced long-term extinction when administered before extinction training. MDMA increased the expression of Fos in the amygdala and medial prefrontal cortex (mPFC), whereas increases in Bdnf expression were observed only in the amygdala after extinction training. Extinction enhancements were recapitulated when MDMA (1 μg) was infused directly into the basolateral complex of the amygdala (BLA), and enhancement was abolished when BDNF signaling was inhibited before extinction. These findings suggest that MDMA enhances fear memory extinction through a BDNF-dependent mechanism, and that MDMA may be a useful adjunct to exposure-based therapies for PTSD and other anxiety disorders characterized by altered fear learning.”

Authors: Matthew B. Young, Raul Andero, Kerry J. Ressler & Leonard L. Howell 

Smmary

INTRODUCTION

Post-traumatic stress disorder (PTSD) is characterized by recurrent and intrusive memories for a traumatic experience. MDMA-assisted psychotherapy improves symptoms of PTSD for years after the intervention, but no preclinical data provide a mechanistic basis for these findings.

The learning and extinction of fear has been studied extensively in rodent models, and is similar to exposure-based therapy. The effect of clinically relevant doses of MDMA on either fear extinction or exposure-based therapy has not been explored to date.

Recovery from PTSD and extinction learning depend on brain-derived neurotrophic factor (BDNF), and MDMA modulates activity in the amygdala and medial prefrontal cortex under resting conditions, but not during fear memory retrieval.

MDMA improves fear extinction in mice by increasing activity in the amygdala and mPFC, and by increasing the expression of BDNF mRNA.20 These effects were abolished when BDNF signaling was disrupted before fear extinction training.

Animals

Mice were bred at the Yerkes National Primate Research Center at the Emory University and maintained on ad libitum food and water. All experiments were performed on 10- to 16-week-old male mice.

Drugs

S,R( )-MDMA was dissolved in saline and injected intraperitoneally. Sheep anti-BDNF antibody was purchased from EMD Millipore and was administered centrally.

Surgeries

Animals were placed under ketamine+dexmedetomidine anesthesia before surgery using a stereotax. Guide cannulae were implanted for intravenous injection of intraperitoneal atipamezole and meloxicam, and mice recovered for at least 4 days before experimentation.

IL and BLA infusions

Mice were habituated to the investigator and the infusion procedure in two 4-min handling sessions followed by 3 min of mock infusion. MDMA or anti-BDNF antibody was infused bilaterally into IL, BLA or adjacent regions 10 min before extinction training.

Fear conditioning and extinction

Mice were exposed to cued fear conditioning, fear extinction training and extinction testing. After habituation, mice were placed in individual plastic buckets with bedding and lids for 30 – 60 min.

Cued fear conditioning consisted of a single pairing of a CS tone and a US footshock, and extinction training consisted of a suboptimal regimen of four CS re-exposures separated by 45 s.

Mice were exposed to CSs and tested for freezing response 1 or 10 days later. Extinction training improved freezing response.

All experiments were performed with eight animals per group. Subjects were pseudorandomly assigned to groups to account for shock sensitivity.

Locomotor behavior

Experiments were carried out in 40 x 40 x 30 cm photocell cages with 32 photocells positioned 2.5 cm off the cage floor. MDMA or saline were used to treat animals.

mRNA was taken from 1-mm diameter brain punches of the mPFC and amygdala. The mRNA was isolated and purified from tissue punches using the RNeasy Mini Kit (catalog 74106, Qiagen) and reverse-transcribed with the RT2 First Strand Kit (catalog 330401, Qiagen).

Statistical analysis

Behavioral data were analyzed with SPSS 22.0 and Prism 5.0 and compared with saline-treated controls using Dunnett’s test and Bonferroni’s test. The data variance was similar between the groups compared in each statistical analysis.

extinction training

MDMA reduced freezing to the CS tone during the extinction training session and reduced freezing to the CS tone 24 hours later. MDMA increased locomotor behavior but did not alter freezing in the extinction apparatus.

We tested whether MDMA’s effect on fear extinction persisted in response to manipulations known to interfere with fear extinction. MDMA-induced fear extinction enhancements persisted for at least 10 days.

MDMA decreased freezing during the initial trial, but not the following day, suggesting that MDMA did not impair reconsolidation. MDMA also decreased freezing during the first six tones of a 14-tone trial, but returned to control levels by the end of the trial.

MDMA treatment that enhances extinction increases neurobiological markers of extinction learning

We measured changes in expression in the early-response gene Fos in response to MDMA and/or extinction training, and found that MDMA increased levels of Fos mRNA in both the mPFC and amygdala 97 min after administration, regardless of whether animals were exposed to extinction training.

MDMA enhances fear extinction via the IL cortex and basolateral amygdala

The amygdala and mPFC have a number of subregions with unique behavioral functions. MDMA exerts its behavioral effects in these subregions.

Bilateral infusions of MDMA into the IL or BLA 10 min before extinction training did not reduce conditioned freezing to the CS tone during the extinction training session.

Extinction enhancements by MDMA in the BLA require intact BDNF signaling

To determine if extinction enhancements mediated by MDMA in the BLA depend on BDNF there, we obstructed BDNF signaling before extinction by directly infusing a BDNF-neutralizing antibody. The anti-BDNF treatment did not alter the reductions in freezing induced by 7.8 mg kg -1 of MDMA during extinction training.

DISCUSSION

MDMA treatment before extinction training induced long-term reductions in conditioned fear, and this effect persisted even when the fear-eliciting stimulus was presented in a novel context. MDMA enhanced extinction learning in a BDNF-dependent manner.

MDMA reduced conditioned freezing during extinction testing, whether animals were initially conditioned with either a single CS – US pairing or multiple CS – US pairings. These reductions were due to MDMA’s psychostimulant properties, and not to an effect on extinction acquisition.

MDMA treatment and extinction reduced conditioned freezing not only the day after treatment, but for several days afterward. This suggests that MDMA induces a powerful form of extinction learning.

Our results suggest that MDMA enhances extinction learning only when combined with extinction training, and that this effect is due to an effect between MDMA and activity-dependent processes in the amygdala that drive subsequent BDNF signaling important for consolidation.

Acutely administered MDMA increases BDNF expression in the mPFC 24 h later. However, the effect of systemically administered MDMA was abolished by disrupting BDNF signaling in the BLA.

MDMA’s neuropharmacological effects are wide-ranging, and it is difficult to speculate on how it might rapidly increase extinction learning and Bdnf expression. However, MDMA increases serotonin and norepinephrine levels and binds directly to 5-HT2A receptors, which may be why MDMA enhances fear memory extinction.

MDMA was explored as an adjunct to psychotherapy in the 1970s, and it is likely that MDMA increases BDNF signaling in the amygdala, which enhances fear extinction learning in PTSD patients.

ACKNOWLEDGMENTS

This research was conducted at the Yerkes National Primate Research Center and complied with all laws of the United States of America.