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

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.

Keywords: Fear extinction; Fear-potentiated startle; MDMA; Monoamines; Serotonin.

Fig 1. Effect of transporter inhibitors on fear extinction and MDMA-induced enhancement of fear extinction

(a-c; left) Conditioned freezing during extinction training 60 min after receiving a dose of citalopram, reboxetine or RTI-336. (a-c-right) Conditioned freezing the following day (n=8/group). (d) Total average conditioned freezing during extinction training in vehicle- or MDMA-treated mice that received vehicle, citalopram, reboxetine, or RTI-336 60 min prior to extinction training. (n=15 in vehicle pre-treatment groups; n=9 in drug pre-treatment groups) (d) Total average conditioned freezing the day after treatment. *p<0.05; ^p<0.01, #p<0.001.

Fig 2. Chronically-administered citalopram inhibits MDMA's effect on fear extinction 48 h later

(a) Experimental model. (b) Conditioned freezing across groups of 4 CS re-exposures during extinction training (left) and testing (right) (n=10/group). (c) Average total conditioned freezing to CS during extinction training. Chronically administered citalopram blocked MDMA-induced reductions in conditioned freezing. (d) Average total conditioned freezing to CS during extinction testing. Chronically-administered citalopram significantly obstructed MDMA-induced reductions in conditioned freezing. (e) Experimental model. (f) Average total conditioned freezing during extinction testing. Acute citalopram treatment 24 h prior to extinction training did not alter MDMA‟s lasting effect on conditioned freezing during extinction testing (n=8/group). *p < 0.05; # p < 0.001

Fig 3. Effect of fenfluramine and the 5-HT_2A receptor on fear memory extinction

(a) Total conditioned freezing to CS re-exposure during extinction training (left) and testing (right) when fenfluramine was administered 30 min prior to extinction training (n=8/group). (b) Total number of head-twitches induced by the 5-HT_2AR agonist DOI (1 mg/kg) 48 h after 22 days of daily vehicle or citalopram administration (n=5/group). (c) Total conditioned freezing to CS re-exposure during extinction training when the 5-HT_2AR antagonist M100 was administered 30 min prior to MDMA treatment (n=8/group). (d) Total conditioned freezing to CS re-exposure during extinction testing the day after extinction training (n=8/group). *p < 0.05; ^p < 0.01; #p < 0.001

Fig 4. MDMA's effects on extinction of fear-potentiated startle (FPS)

(a) Experimental model. MDMA was administered 20 min prior to extinction training. (b) Average FPS across sets of 4 CS re-exposures during memory testing (Pre), extinction training (Day 7), and extinction testing (Day 8). 7.8 mg/kg treatment resulted in significantly less FPS during CS trials 1,2, 5 and 10 compared to vehicle treatment (n=16/group). (c) Total FPS in response to CS re-exposure during extinction training when MDMA was administered 20 min prior to extinction training. All treatment groups had significantly less FPS than the control vehicle-treated group (n=16/group). (d) Total FPS in response to CS re-exposure during extinction testing. Only 7.8 mg/kg treatment resulted in significantly less FPS than the vehicle-treated group (n=16/group). *p < 0.05; ^p < 0.01; #p < 0.001.

Fig 5. Chronically-administered citalopram inhibits MDMA's effect on the extinction of FPS

(a) Experimental model. (b) Average FPS across sets of 4 CS re-exposures during memory testing (Pre), extinction training (Day 25), and extinction testing (Day 26) when MDMA (7.8 mg/kg) was administered 20 min prior to extinction training (n=12/group). (c) Total FPS in response to CS re-exposure during extinction training when MDMA or vehicle was administered 20 min prior to extinction training. Both groups treated with MDMA exhibited significantly less FPS then vehicle-treated groups regardless of chronic treatment. (d) Total FPS in response to CS re-exposure during extinction testing. *p < 0.05; ^p < 0.01; #p < 0.001.