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.
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.
Papers cited by this study that are also in Blossom
Berman, R. M., Cappiello, A., Anand, A. et al. · Biological Psychiatry (2000)
Feder, A., Parides, M. K., Murrough, J. W. · JAMA Psychiatry (2014)
Mcghee, L. L., Maani, C. V., Garza, T. H. et al. · Journal of Trauma Injury Infection and Critical Care (2008)
Papers in Blossom that reference this study
Meinhardt, M., Skorodumov, I., Walter, F. et al. · Research Square (2026)
PTSD is a chronic disorder characterised by persistent re-experiencing, avoidance, emotional numbing and hyperarousal; deficits in extinction of conditioned fear are implicated in its persistence. Earlier research has established that Pavlovian fear conditioning and extinction depend on interactions among the amygdala, hippocampus and medial prefrontal cortex (mPFC), and that glutamatergic signalling plays a central role in the formation and treatment of traumatic memories. Conventional antidepressants have limited speed and efficacy for PTSD, while recent clinical studies indicate that ketamine, an NMDA receptor antagonist, produces rapid symptomatic improvement in PTSD and induces fast synaptic and antidepressant effects in preclinical models via mTORC1-dependent increases in synapse number and function in mPFC. Girgenti and colleagues set out to determine whether a single systemic dose of ketamine enhances extinction learning and its recall in a rat model of auditory fear conditioning and extinction, and to identify the molecular mechanisms involved. Specifically, the study tests whether ketamine administered after conditioning improves extinction and reduces fear renewal, whether ketamine plus extinction activates mTORC1 signalling and neuronal activity in the mPFC, and whether mTORC1 and AMPA receptor activity are required for ketamine's effects.
Male Sprague-Dawley rats (7–9 weeks, 175–250 g) were used and housed under standard conditions. Ketamine hydrochloride was given systemically at 10 mg/kg i.p. (diluted to 10 mg/ml), rapamycin was dissolved in DMSO and infused locally into mPFC (0.2 nmol in 0.2 μl), and the AMPA antagonist NBQX was given systemically at 10 mg/kg i.p. Surgery placed bilateral guide cannulas targeting the interface of prelimbic and infralimbic mPFC (coordinates reported as −0.9 mm AP, ±1.5 mm ML, −3.3 mm below dura, 30° angle); animals recovered 7–9 days before behavioural procedures. Fear conditioning occurred in context A with seven CS–US pairings (30 s tone co-terminating with a 1 s, 0.66 mA footshock). Twenty-four hours later rats received ketamine or saline and, 24 hours after drug treatment, were placed in a modified context B for extinction training: 12 non‑reinforced CS presentations per day for 4 consecutive days (3 min acclimation, inter-tone intervals 60–90 s). Spontaneous recovery and renewal were tested one week after extinction by presenting a single CS in context B and then context A. Freezing during CS presentation (percentage of time) was measured using an automated video system validated against blind hand scoring (correlation >0.90). Molecular assays: tissue for biochemical analysis (mPFC and amygdala) was collected 90 min after the last tone on extinction day 2. Synaptoneurosome-enriched extracts and crude nuclear extracts were prepared for Western blotting of phosphorylated and total mTOR, p70S6K, ERK, Akt, and cFos, with quantification normalised to total protein levels. Rapamycin or vehicle infusions were administered 30 min before systemic ketamine or saline to test necessity of mTORC1 signalling. In a separate cohort, NBQX was given 10 min before ketamine to test AMPA receptor involvement. Statistics: group comparisons used Student's t-tests for two-group biochemical data, two-way ANOVA for four-group comparisons, and repeated-measures ANOVA for extinction experiments; Bonferroni post hoc tests were applied where appropriate. Sample sizes for behavioural cohorts were reported as n = 16–19 for the main extinction experiment, molecular n = 6 in many assays, rapamycin cohorts n = 6, and NBQX cohort n = 10; the paper notes sample sizes were based on prior work but does not present a priori power calculations in the extracted text.
Behavioural effects: Rats given a single 10 mg/kg i.p. dose of ketamine 24 hours after conditioning showed no difference from saline on the first day of extinction, but exhibited significantly reduced freezing on the second day of extinction (repeated-measures ANOVA, treatment effect F(18,70) = 6.255, p < 0.05). This reduction persisted into the third day (Block 1 treatment effect F(1,70) = 6.255, post hoc p < 0.01). One week after extinction, there was no significant difference in spontaneous recovery when tested in the extinction context, but ketamine-treated animals demonstrated significantly reduced fear renewal when returned to the original training context plus cue (t = 2.897, df = 18, p < 0.05). Freezing elicited by exposure to context alone did not differ between groups, suggesting the reduction in renewal required cue plus context. mTORC1 and upstream kinases: Analysis of synaptoneurosome-enriched mPFC extracts collected after extinction day 2 revealed that ketamine plus extinction produced a robust ~two-fold increase in phosphorylated p70S6K compared with saline plus extinction (t = 2.32, df = 10, p < 0.05), while phosphorylated mTOR itself was not significantly changed (t = 1.092, df = 10, p = 0.30). Upstream signalling showed significant increases in phosphorylated ERK (approximately two-fold; t = 3.11, df = 10, p < 0.05) and a smaller but significant increase in phosphorylated Akt (t = 4.171, df = 10, p < 0.01), indicating activation of kinases that converge on mTORC1. Requirement for mTORC1: Local infusion of rapamycin into the mPFC 30 min before ketamine blocked the biochemical induction of p-p70S6K: ketamine + DMSO increased p-p70S6K versus saline + DMSO (t = 10.16, df = 4, p < 0.001), and rapamycin prevented that increase (ketamine + rapamycin vs ketamine + DMSO, t = 3.84, df = 4, p < 0.01). Behaviourally, rapamycin infusion completely abolished ketamine's enhancement of extinction recall on the second day (RM ANOVA treatment effect F(3,100) = 37.01, p < 0.0001; post hoc ketamine vs ketamine + rapamycin p < 0.01 for multiple blocks). The authors note a cohort-specific decrease in freezing on day 1 in the cannulated animals that might relate to surgical stress. Neuronal activity and AMPA dependence: Ketamine plus extinction increased cFos protein in the mPFC 90 min after extinction day 2 (t = 3.66, df = 10, p < 0.01, n = 6); cFos in the amygdala trended downward but did not reach significance (t = 2.36, df = 10, p = 0.06). Pretreatment with the AMPA antagonist NBQX (10 mg/kg, i.p.) 10 min before ketamine produced a partial but significant blockade of ketamine's enhancement of extinction (RM ANOVA F(2,132) = 5.574, p < 0.05; post hoc ketamine vs NBQX + ketamine p < 0.05 for blocks 2 and 3), indicating AMPA receptor-dependent mechanisms contribute to the effect.
Girgenti and colleagues interpret their findings as evidence that a single systemic dose of ketamine administered after fear conditioning can accelerate extinction learning and reduce later fear renewal, effects that persist for at least one week. They link these behavioural outcomes to activation of an mTORC1-dependent signalling cascade and increased neuronal activity in the mPFC, as indicated by elevated p‑p70S6K, upstream activation of ERK and Akt, and increased cFos. The requirement for mTORC1 signalling was directly tested and supported by intra-mPFC rapamycin infusions that blocked both biochemical and behavioural effects. Similarly, partial blockade by an AMPA receptor antagonist supports a role for glutamate-AMPA receptor activity in ketamine's enhancement of extinction. The authors position these results within existing work showing that ketamine rapidly increases synapse number and function in mPFC and that mPFC–amygdala circuitry is central to extinction. They suggest the mechanism may involve ketamine-induced disinhibition of GABA interneurons, producing a glutamate burst that strengthens infralimbic mPFC synapses and enhances inhibitory control over amygdala outputs that mediate fear. However, the paper acknowledges alternative interpretations that cannot be excluded from the current data, notably that ketamine might impair reconsolidation of the original fear memory rather than selectively enhancing extinction. Key limitations and uncertainties noted by the investigators include cohort differences possibly related to surgical stress in cannulated animals and the inability of the present experiments to fully dissociate effects on reconsolidation versus extinction consolidation. The authors call for further studies to dissect the specific contributions of infralimbic versus prelimbic mPFC subregions and to test translational potential. Clinically, they frame the findings as supporting development of ketamine-like glutamatergic agents with improved safety profiles as potential rapid-acting treatments to augment extinction-based therapies for PTSD and related fear disorders, while recognising that more work is required to translate these mechanisms to humans.
The study concludes that a single dose of ketamine can produce rapid and sustained enhancement of fear extinction in rats, and that these behavioural effects depend on activity‑dependent mTORC1 signalling and synaptic mechanisms in the mPFC. By showing that intra-mPFC rapamycin blocks both biochemical markers and behavioural enhancement, and that AMPA receptor blockade partially attenuates the effect, the authors argue that ketamine's capacity to facilitate extinction involves glutamate‑driven, mTORC1‑mediated synaptic changes. They suggest these mechanisms may underlie ketamine's therapeutic potential for PTSD and advocate for the development of novel ketamine-like agents with fewer side effects as candidate adjuncts to extinction-based therapies.
Statistics were performed using GraphPad Prism 6.05 for OSX. All data are expressed as the mean ± SEM. Sample sizes were based on previous behavioral and biochemical studies conducted in our laboratory. Data for signaling proteins with two groups were analyzed by Student's t-test and for four groups by two way ANOVA. For all extinction experiments repeated-measures ANOVA were used. Significant group differences were only reported if protected by significant effects or interactions with ANOVA. In all cases, if a statistically significant interaction was found, additional comparisons were calculated. Bonferroni post hoc analysis was performed where appropriate.
The rapid-acting antidepressant effects of ketamine in rodent models and clinical trials have been well established, and recent studies demonstrate the efficacy of ketamine for the treatment of PTSD. Blockade of NMDA receptors by ketamine increases the number and function of synapses in the mPFC, a region that also plays a critical role in the acquisition and retrieval of extinction. Our findings demonstrate that a single dose of ketamine enhances the recall of extinction learning. It is also possible that ketamine inhibits reconsolidation of fear memory and thereby reduces freezing during extinction training, a possibility that cannot be ruled out at the present time. On day 2 of extinction training we observe a large change in freezing levels between the ketamine and saline treated animals. This observation suggests the possibility that ketamine enhances extinction memory. Moreover, fear renewal was decreased in ketamine treated animals 7 days later. These findings demonstrate a rapid and long-lasting enhancement of fear extinction that could be mediated by ketamine stimulation of mTORC1 signaling pathways and sustained synaptic actions in the mPFC. Mechanistic studies demonstrate that ketamine activates the mTORC1 cascade in the mPFC and that the synaptic and antidepressant behavioral responses are dependent on mTORC1 signaling (i.e blocked by rapamycin). We found that extinction in animals receiving ketamine increased the phosphorylated and activated form of p70S6K in the mPFC, as well as levels of the upstream kinases p-ERK and p-Akt. A role for mTORC1 was directly tested by infusions of rapamycin into the mPFC prior to ketamine administration, which completely blocked ketamine enhancement of extinction. Previous studies demonstrate that ketamine increases the number and function of spine synapses in layer V pyramidal neurons in the mPFC and that these effects are also dependent on mTORC1 signaling. Together these findings support the hypothesis that ketamine's enhancement of extinction results from increased synaptic function in the mPFC. Increased synaptic number and function could enhance synaptic connectivity of mPFC with target regions that control fear conditioning and extinction such as the amygdala. We also found that ketamine administration increases levels of cFos in the mPFC after extinction training, indicating an increase in neuronal activation during the enhancement of extinction training. Previous studies demonstrate that ketamine increases glutamate release in the mPFCand increases levels of cFos, presumably via disinhibition of GABA interneuron activity. Previous reports also indicate that the rapid antidepressant actions of ketamine require activation of glutamate AMPA receptors. In the current study we observed that pretreatment with the AMPA receptor antagonist NBQX partially blocked ketamine-induced enhancement of extinction. Together these studies indicate a role for glutamate-AMPA receptor activity in the actions of ketamine on fear extinction. Several lines of evidence suggest that glutamatergic neurotransmission plays a critical role in the pathogenesis of anxiety and fear disorders. Fear extinction involves neurocircuitry of at least three main brain areas: the amygdala, mPFC, and the hippocampus. Within the mPFC, afferents from glutamatergic pyramidal neurons in the infralimbic subregion activate glutamatergic neurons in the basolateral nucleus of the amygdala (BLA) that synapse onto inhibitory GABAergic interneurons. This connection is thought to gate signaling from the BLA to the central nucleus of the amygdala. In this circuit, fear extinction overrides fear conditioning via strengthening of those synapses. This inhibitory drive is powerful enough to overcome the excitatory responses associated with a pre-existing fear conditioning circuit. It is possible that the glutamate burst caused by ketamine and the subsequent increased synaptic connectivity strengthens these infralimbic mPFC synapses resulting in enhancement of fear extinction. Future studies will be required to test the role of infralimbic mPFC in the enhancement of fear extinction by ketamine, and to differentiate effects on prelimbic mPFC, which has been implicated in the formation of fear conditioning. There are currently only two FDA approved drug treatments for PTSD, the SSRI's paroxetine and sertraline, highlighting the need for additional pharmacotherapies for this disorder. As with most classical antidepressants, the SSRIs require several weeks of treatment to produce a therapeutic response, and even then have limited efficacy for the treatment of PTSD symptoms. Several lines of clinical evidence support the therapeutic potential of glutamatergic agents. Soldiers with severe burns that had received perioperative ketamine during hospitalization were found to have lower incidence of developing PTSD. Moreover, a recent clinical trial demonstrated that ketamine infusion was associated with significant and rapid reduction in PTSD symptoms when compared to midazolam. Although d-cycloserine (DCS), a partial NMDA agonist, was ineffective for reducing fear relapse, it was found to augment fear extinction learning in rodents and humans. DCS had mixed efficacy for reducing PTSD symptoms in a randomized placebo-controlled study. These findings indicate that glutamate NMDA receptor blocking agents have potential for treating PTSD and related disorders, and the current study provides evidence that this may occur in part via activity-dependent mTORC1 signaling in the mPFC. Development of novel ketamine-like agents with fewer side effects is a major focus of antidepressant drug development efforts and could also provide new, safer and more effective treatments for PTSD and other fear-related disorders.
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