Ketamine accelerates fear extinction via mTORC1 signaling

This rat study explores the mechanisms underlying the actions of ketamine in fear conditioning, extinction, and renewal. The study found that ketamine produces long-lasting positive changes in fear extinction, through mTORC1/protein synthesis in the medial prefrontal cortex. This offers more evidence for why and how ketamine may be a potential approach for the treatment of PTSD.

Abstract

“Impaired fear extinction contributes to the persistence of post-traumatic stress disorder (PTSD), and can be utilized for the study of novel therapeutic agents. Glutamate plays an important role in the formation of traumatic memories, and in the pathophysiology and treatment of PTSD, highlighting several possible drug targets. Recent clinical studies demonstrate that infusion of ketamine, a glutamate NMDA receptor antagonist, rapidly and significantly reduces symptom severity in PTSD patients. In the present study, we examine the mechanisms underlying the actions of ketamine in a rodent model of fear conditioning, extinction, and renewal. Rats received ketamine or saline 24 h after fear conditioning and were then subjected to extinction-training on each of the following three days. Ketamine administration enhanced extinction on the second day of training (i.e., reduced freezing behavior to cue) and produced a long-lasting reduction in freezing on exposure to cue plus context 8 days later. Additionally, ketamine and extinction exposure increased levels of mTORC1 in the medial prefrontal cortex (mPFC), a region involved in the acquisition and retrieval of extinction, and infusion of the selective mTORC1 inhibitor rapamycin into the mPFC blocked the effects of ketamine on extinction. Ketamine plus extinction also increased cFos in the mPFC and administration of a glutamate-AMPA receptor antagonist blocked the effects of ketamine. These results support the hypothesis that ketamine produces long-lasting mTORC1/protein synthesis and activity dependent effects on neuronal circuits that enhance the expression of extinction and could represent a novel approach for the treatment of PTSD.”

Authors: Matthew J. Girgenti, Sriparna Ghosal, Dora LoPresto, Jane R. Taylor & Ronald S. Duman

Summary

Ketamine, a glutamate NMDA receptor antagonist, enhances fear extinction in a rodent model of fear conditioning, extinction, and renewal, and increases levels of mTORC1 and cFos in the medial prefrontal cortex. Ketamine could represent a novel approach for the treatment of post-traumatic stress disorder.

1. Introduction

Post-traumatic stress disorder (PTSD) is a chronic and debilitating disorder characterized by persistent re-experiencing of memories, avoidance of cues or situations that are reminiscent of the traumatic event, emotional numbing, and hyperarousal.

Significant progress has been made in understanding the neurobiological basis of fear, and animal models of fear conditioning and extinction learning represent an ideal paradigm for preclinical assessments of PTSD and for identifying novel pharmacotherapies.

Antidepressants, particularly selective serotonin reuptake inhibitors, can reduce PTSD symptoms in humans and fear in rodents when combined with extinction therapy. However, currently available antidepressants have several limitations, including slow onset of action and low rates of efficacy.

There is mounting evidence that glutamate plays a role in stress responsiveness, traumatic memories, and the pathophysiology of PTSD. Ketamine, a glutamate N-methyl-D-aspartate receptor antagonist, is effective in treating PTSD.

Ketamine increases synapse number and function in the mPFC and the mTORC1 signaling pathway, which is involved in translation and synaptic protein synthesis. These actions could explain why ketamine improves fear extinction in adult rats.

Male Sprague-Dawley rats were used for all experiments. They were kept under standard conditions with a 12-h light/dark cycle and food and water were available ad libitum.

Ketamine hydrochloride, rapamycin, and NBQX were diluted to 10 mg/ml in 0.9% saline and administered by i.p. injection.

2.3. Apparatus

Fear conditioning was carried out in four identical operant chambers constructed from aluminum and Plexiglas. Each chamber contained a grid floor and a waste pan, and was equipped with a miniature high-speed Firewire monochromatic camera.

For extinction training, the context was modified by using a smooth white acrylic insert and a black plastic triangular insert. The house lights were kept off and a novel odor was used.

2.4. Fear conditioning procedure

Rats were placed in the training context and received seven pairings of the CS and US. The time-spent freezing during delivery of the CS tone was scored and the next day they received a single injection of ketamine or saline.

Twenty-four hours after drug treatment, rats were placed in context B and received 12 non-reinforced presentations of the CS. This procedure was repeated over the next 3 days to produce 4 separate days of extinction training.

Freezing was measured using an automated computer analysis system and by blind hand scoring. A correlation of >0.90 was found between the automated system and human observer values.

2.5. Surgery and drug infusions

Rats were anesthetized with a ketamine-xylazine cocktail and placed into a stereotaxic apparatus. Bilateral guide cannulas were inserted into the medial prefrontal cortex as described previously.

Rats were fear conditioned and then given rapamycin or DMSO in the mPFC 24 hours after conditioning. The rapamycin was left in place for 3 min before ketamine or saline injections and animals with incorrect placement were eliminated from the behavioral analysis.

2.6. Western blotting

Brain tissue was collected 90 min after the last tone was presented during the extinction recall test on day 2. The amygdala and medial prefrontal was carefully dissected and frozen on dry ice and stored at 80 °C until processing. After incubation, membranes were washed in TBS-T and incubated with an appropriate peroxidase-labeled secondary antibody (1:10,000; Vector Laboratories). Bands were visualized with enhanced chemiluminescence and exposed to Hyblot CL autoradiography film.

The intensity of the protein bands was quantified using image analysis software, and the values were normalized to the average signal for the total protein levels.

2.7. Statistics

Statistics were performed using GraphPad Prism 6.05 for OSX and Student’s t-test and two way ANOVA were used for all analyses. Bonferroni post hoc analysis was performed where appropriate.

3.1. Ketamine enhances fear extinction learning

Sprague-Dawley rats were fear conditioned using 7 pairs of a neutral tone that co-terminated with an aversive stimulus. Twenty-four hours after fear conditioning, the rats were administered ketamine or saline. The next day, the animals were subjected to extinction training in a different context from the fear conditioning for 3 consecutive days. The rats receiving ketamine showed a stronger extinction memory compared to the saline group.

Ketamine enhanced the recall of fear extinction as well as reduced the recovery of fear responses following extinction training. Ketamine treatment was effective at 8 days after the last day of extinction training.

3.2. Ketamine plus fear extinction increases mTORC1 signaling in the mPFC

Ketamine increased mTORC1 signaling in the mPFC, and p70S6K was the most significant increase. There was no significant difference in p-mTOR levels between ketamine and saline plus extinction training.

Ketamine plus extinction training increased the phosphorylated and activated forms of ERK and Akt in the mPFC, suggesting that these pathways are involved in the actions of ketamine plus extinction training.

Ketamine’s rapid synaptic and antidepressant actions require mTORC1 signaling. Rapamycin blocked ketamine’s induction of p70S6 Kinase in the mPFC, and ketamine’s effect on fear extinction was completely blocked by rapamycin.

We found that rapamycin completely blocked ketamine enhancement of extinction recall on the second day and that rapamycin blocked phosphorylation of p70S6 kinase in the mPFC, which is a requirement for mTORC1 signaling in the enhancement of fear extinction learning after ketamine.

3.4. Ketamine enhancement of extinction: induction of cFos and influence of AMPA receptor blockade

We measured levels of cFos in the mPFC and amygdala to determine if ketamine plus extinction influences neural activation of mPFC and amygdala. Our results support earlier findings that increased mPFC activity leads to reduced fear-related amygdala processing that is associated with improved extinction learning and recall.

Ketamine has synaptic and antidepressant actions that require glutamate AMPA receptors. Pretreatment with a selective AMPA receptor inhibitor, 2,3-dihy-droxy-6-nitro-7sulfamoyl-benzol(f)quinoxaline-2,3-dione (NBQX), attenuated the effects of ketamine on fear extinction in rats.

4. Conclusion

Ketamine has been shown to have rapid-acting antidepressant effects in rodent models and clinical trials, and recent studies demonstrate the efficacy of ketamine for the treatment of PTSD. Ketamine increases the number and function of synapses in the mPFC, which is involved in the acquisition and retrieval of extinction learning.

Ketamine enhances extinction by increasing the number and function of spine synapses in layer V pyramidal neurons in the mPFC, and by increasing the phosphorylated and activated form of p70S6K in the mPFC. This increase in synaptic function may be associated with increased synaptic connectivity.

Ketamine administration increases levels of cFos in the mPFC after extinction training, indicating an increase in neuronal activation. Ketamine’s rapid antidepressant actions require activation of glutamate AMPA receptors.

Several lines of evidence suggest that glutamatergic neurotransmission plays a critical role in the pathogenesis of anxiety and fear disorders. Ketamine may enhance fear extinction by strengthening the synapses in the infralimbic mPFC, which are thought to be involved in fear conditioning.

There are currently only two FDA approved drug treatments for PTSD, the SSRI’s paroxetine and sertraline. Several lines of clinical evidence support the therapeutic potential of glutamatergic agents for the treatment of PTSD and related disorders, and the current study provides evidence that this may occur in part via activity-dependent mTORC1 signaling.

Fig. 1.

Ketamine accelerates extinction learning and reduces recovery of fear memory with time. Ketamine-treated rats showed significantly reduced freezing on the second and third blocks of the second session of training. One week after the extinction event, the ketamine-treated rats showed reduced fear induced freezing compared to the saline-treated animals. However, there was no difference in freezing behavior during the first 3 min of fear renewal.

Fig. 2.

Ketamine and fear extinction influence mTORC1 signaling in the mPFC. The phosphorylated and activated forms of mTOR, p70S6K, ERK, and Akt and total levels of each were analyzed by western blot.

Fig. 3.

Inhibition of mTORC1 signaling in the mPFC blocks ketamine’s enhancement of fear extinction. Ketamine increased extinction on both training days, possibly due to stress associated with cannula surgeries, and rapamycin infusion completely blocked this effect.

Fig. 4.

Rats underwent fear conditioning, ketamine administration, and extinction training. The effects of ketamine and fear extinction on cFos expression were examined, and the effect of glutamate AMPA receptor blockade on ketamine-induced enhancement of extinction was examined.