Ketamine accelerates fear extinction via mTORC1 signaling

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.