Inhibition of spontaneous recovery of fear by mGluR5 after prolonged extinction training.

Fear behavior is vital for survival and involves learning contingent associations of non-threatening cues with aversive stimuli. In contrast, excessive levels of fear can be maladaptive and lead to anxiety disorders. Generally, extensive sessions of extinction training correlates with reduced spontaneous recovery. The molecular mechanisms underlying the long-term inhibition of fear recovery following repeated extinction training are not fully understood. Here we show that in rats, prolonged extinction training causes greater reduction in both fear-potentiated startle and spontaneous recovery. This effect was specifically blocked by metabotropic glutamate receptor 5 (mGluR5), but not by mGluR1 antagonists and by a protein synthesis inhibitor. Similar inhibition of memory recovery following prolonged extinction training was also observed in mice. In agreement with the instrumental role of mGluR5 in the prolonged inhibition of fear recovery, we found that FMR1-/- mice which exhibit enhanced mGluR5-mediated signaling exhibit lower spontaneous recovery of fear after extinction training than wild-type littermates. At the molecular level, we discovered that prolonged extinction training reversed the fear conditioning-induced increase in surface expression of GluR1, AMPA/NMDA ratio, postsynaptic density-95 (PSD-95) and synapse-associated protein-97 (SAP97). Accordingly, delivery of Tat-GluR2(3Y), a synthetic peptide that blocks AMPA receptor endocytosis, inhibited prolonged extinction training-induced inhibition of fear recovery. Together, our results demonstrate that prolonged extinction training results in the mGluR5-dependent long-term inhibition of fear recovery. This effect may involve the degradation of original memory and may explain the beneficial effects of prolonged exposure therapy for the treatment of phobias.

GABAA receptor endocytosis in the basolateral amygdala is critical to the reinstatement of fear memory measured by fear-potentiated startle.

Reinstatement represents a phenomenon that may be used to model the effects of retraumatization observed in patients with posttraumatic stress disorder (PTSD). In this study, we found intraperitoneal injection of the ß-adrenergic receptor antagonist propranolol (10 mg/kg) 1 h before reinstatement training attenuated reinstatement of fear memory in rats. Conversely, reinstatement was facilitated by intra-amygdalar administration of ß-adrenergic receptor agonist isoproterenol (Iso; 2 µg per side) 30 min before reinstatement training. The frequency and amplitude of the miniature IPSC (mIPSC) and the surface expression of the ß3 and γ2 subunits of the GABA(A) receptor (GABA(A)R) were significantly lower in reinstated than in extinction rats, whereas the AMPA/NMDA ratio and the surface expression of GluR1 and GluR2 in the amygdala did not differ between groups. In amygdala slices, Iso-induced decrease in the surface ß3 subunit of GABA(A) receptor was blocked by a Tat-conjugated dynamin function-blocking peptide (Tat-P4) pretreatment (10 µm for 30 min). By contrast, Tat-scramble peptide had no effect. Intravenous injection (3 µmol/kg) or intra-amygdalar infusion (30 pmol per side) of Tat-P4 interfered with reinstatement. Reinstatement increased the association between protein phosphatase 2A (PP2A) and the ß3 subunit of the GABA(A)R, which was abolished by PP1/PP2A inhibitors okadaic acid and calyculin A. These results suggest the involvement of ß-adrenergic receptor activation and GABA(A) receptor endocytosis in the amygdala for the reinstatement in fear memory.

Alterations of excitatory transmission in the lateral amygdala during expression and extinction of fear memory.

Understanding the neurophysiology of fear extinction has important implications for the treatment of post-traumatic stress disorders. Here we report that fear conditioning resulted in an increase in AMPA/NMDA ratio as well as depression of paired-pulse facilitation (PPF) in neurons of the lateral nucleus of amygdala. These conditioning-induced changes in synaptic transmission were not affected by extinction training. D-cycloserine (DCS), a partial agonist at the glycine-binding site of the NMDA receptor, facilitated extinction and reversed the increase in AMPA/NMDA ratio without altering the depression of PPF when administered before extinction training. Extinction training, however, significantly increased the frequency and amplitude of miniature inhibitory post-synaptic currents and these effects were unaffected by the DCS treatment. Disruption of AMPA receptor endocytosis with a synthetic peptide containing a short C-terminal sequence of GluR2 (869YKEGYNVYG877, GluR23Y) specifically blocked DCS-induced reversal of AMPA/NMDA ratio and the facilitation of extinction. These results suggest that extinction training mainly increases inhibitory transmission leaving conditioning-induced excitatory association unaltered. DCS does not affect inhibitory transmission but reverses the conditioning-induced post-synaptic memory trace when administered before extinction training.

Block of gamma-aminobutyric acid-A receptor insertion in the amygdala impairs extinction of conditioned fear.

BACKGROUND: Extinction is a complex phenomenon but generally is regarded as a new inhibitory learning that suppresses the original memory. However, how or from where the inhibition originates remains to be determined. In the present study, we examine whether increase in the expression of gamma-aminobutyric acid (GABA)(A) receptors in the amygdala is required for extinction by employing cell-permeable TAT-conjugated peptide (TAT)-GABA receptor-associated protein (GABARAP) inhibitory peptide to block GABA(A) receptor insertion. METHODS: Retention of fear memory was assessed with fear-potentiated startle paradigm. Whole cell patch clamp recordings were performed to record miniature inhibitory postsynaptic current (mIPSC). Western blotting analysis was used to measure the expression of gephyrin, beta2, and gamma2 subunits of GABA(A) receptor. RESULTS: Fear conditioning decreased frequency and amplitude of mIPSC and surface protein levels of beta2 and gamma2 subunits of GABA(A) receptor. Extinction training, by contrast, reversed the decreased frequency and amplitude of mIPSC and surface protein levels of gephyrin and beta2 subunit of GABA(A) receptor. Disruption of GABARAP-GABA(A) receptor interaction in the amygdala with GABARAP inhibitory peptide blocked N-methyl-D-aspartate-mediated GABA(A) receptor insertion in the amygdala. Importantly, it also blocked extinction-induced increase in the frequency and amplitude of mIPSCs, and the reduction of fear-potentiated startle. CONCLUSIONS: GABA(A) receptor insertion in the amygdala contributes a significant part to the extinction of fear memory.

Augmentation of fear extinction by infusion of glycine transporter blockers into the amygdala.

It is known that fear extinction is blocked by the N-methyl D-aspartate (NMDA) receptor antagonist. In this study, we investigate whether extinction could be facilitated by the enhancement of NMDA response, achieved by the blocking of glycine transporters. In amygdala slices, NMDA at a concentration that normally does not have a long-term effect was found to reduce the cellular levels of postsynaptic density protein 95 and synapse-associated protein 97, in addition to the surface expression of GluR1/2, in the presence of a glycine transporter blocker, N[3-(4-fluorophenil)-3-(4'-phenilphenoxy)] propylsarcosine (NFPS). In in vivo experiments, extinction training applied 24 h after conditioning reduced startle potentiation without influencing the conditioning-induced increase in the surface expression of GluR1/2. However, NFPS augmented extinction and reversed the conditioning-induced increase in GluR1/2 when infused bilaterally into the amygdala before extinction training. The effects of NFPS were therefore blocked by the NMDA antagonist. In parallel, NFPS treatment in conjunction with extinction reversed the conditioning-induced alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/NMDA ratio. In behavioral tests, Tat-GluR2(3Y), a synthetic peptide that has been shown to block AMPA receptor endocytosis, inhibited only the additional reduction caused by NFPS treatment, rather than returning the fear potentiation levels to those of fear-conditioned animals that did not undergo extinction. These results suggest that NFPS in combination with extinction training reverses GluR1/2 surface expression and thus augments the extinction of conditioned fear.

The role of prefrontal cortex CB1 receptors in the modulation of fear memory.

Understanding the mechanism of how fear memory can be extinguished could provide potential therapeutic strategies for the treatment of posttraumatic stress disorders. Here we show that infusion of CB1 receptor antagonist into the infralimbic (IL) subregion of the medial prefrontal cortex (mPFC) retarded cue-alone-induced reduction of fear-potentiated startle. Conversely, cannabinoid agonist WIN55212-2 (WIN) facilitated the extinction. Unexpectedly, administration of WIN without cue-alone trials reduced startle potentiation in a dose-dependent manner. The effect of cannabinoid agonists was mimicked by endocannabinoid uptake or fatty acid amide hydrolase inhibitors. Rats were trained with 10 conditioned stimulus (CS(+)) (yellow light)-shock pairings. Extinction training with CS(+) (yellow light)-alone but not CS(-) (blue light)-alone trials decreased fear-potentiated startle. Intra-IL infusion of WIN before CS(-)-alone trials decreased startle potentiation, suggesting that the cannabinoid agonist decreased conditioned fear irrespective of whether the rats underwent CS(+)- or CS(-)-alone trials. Cannabinoid agonists activated extracellular signal-regulated kinases (ERKs) in mPFC slices, and ERK inhibitor blocked the effect of cannabinoid agonists on fear-potentiated startle. These results suggest that CB1 receptors acting through the phosphorylation of ERK are involved not only in the extinction of conditioned fear but also in the adaptation to aversive situations in general.

Chronic cannabinoid administration in vivo compromises extinction of fear memory.

Endocannabinoids are critically involved in the extinction of fear memory. Here we examined the effects of repeated cannabinoid administration on the extinction of fear memory in rats and on inhibitory synaptic transmission in medial prefrontal cortex (mPFC) slices. Rats were treated with the CB1 receptor agonist WIN55212-2 (WIN 10 mg/kg, i.p.) once per day for 7 d. On day 8, the rats were submitted to a standard fear conditioning procedure, and retention of memory was measured with potentiated startle paradigm. We found that (1) WIN-pretreated rats exhibited much less extinction to cue alone presentations; (2) the reduction of fear-potentiated startle normally seen when the CB1 receptor agonists were infused into the mPFC was absent in the WIN-pretreated rats; (3) WIN-induced inhibition of GABAergic transmission was significantly less in slices from the WIN-pretreated rats than that from the vehicle-pretreated control; (4) WIN failed to induce extracellular signal-regulated kinases (ERKs) phosphorylation in the WIN-pretreated rats; and (5) the level of CB1 receptor in the WIN-pretreated rats was lower than that of vehicle-pretreated rats. These results suggest that endocannabinoids within the mPFC play an important role in the extinction of conditioned fear. However, long-term marijuana use may limit its clinical efficacy for the treatment of anxiety disorders.

Augmentation of fear extinction by D-cycloserine is blocked by proteasome inhibitors.

D-Cycloserine (DCS) has been shown to facilitate extinction of conditioned fear in rats and to improve fear reduction of social phobia and fear of heights in human studies. Here, we investigate the mechanism of DCS effect by measuring internalized GluR1 and GluR2 using cell-surface biotinylation techniques. DCS selectively increased NMDA receptor-mediated synaptic response without affecting AMPA receptor-mediated synaptic response. Low-frequency stimulation (LFS) when applied in the presence of DCS induced GluR1 and GluR2 internalization in the amygdala slices. Proteasome inhibitors block DCS facilitation of LFS-induced depotentiation and a reduction in surface levels of GluR1 and GluR2. Furthermore, DCS in combination with LFS reduced cellular levels of PSD-95 and synapse-associated protein 97 (SAP97), which were also blocked by proteasome inhibitors. In the in vivo experiments, DCS-induced reduction of fear-potentiated startle and reversal of conditioning-induced increase in surface expression of GluR1 were blocked by proteasome inhibitors. DCS-treated rats fail to exhibit reinstatement after US-alone presentations. These results suggest that DCS facilitates receptor internalization in the presence of extinction training, resulting in augmented reduction of startle potentiation.

Extinction training in conjunction with a partial agonist of the glycine site on the NMDA receptor erases memory trace.

Much evidence indicates that extinction training does not erase memory traces but instead forms inhibitory learning that prevents the expression of original memory. Fear conditioning induces long-term potentiation and drives synaptic insertion of AMPA receptors into the amygdala. Here we show that extinction training applied 1 h after training reversed the conditioning-induced increase in surface glutamate receptor subunit 1 (GluR1) in parallel with the inhibition of startle potentiation. However, if applied 24 h after training, extinction training reduced startle potentiation without influencing the GluR1 increase. We infused D-cycloserine (DCS), a partial agonist of the glycine site on the NMDA receptor, bilaterally into the amygdala 30 min before extinction training. This augmented the extinction training-elicited reduction in startle and reversed the conditioning-induced increase in GluR1. Delivery of five sets of tetanic stimulation (TS) to the external capsule produced a robust enhancement of synaptic responses in the lateral amygdala neurons that persisted for >2 h. Low-frequency stimulation applied 1 h after TS had no long-lasting effect on synaptic responses. The same treatments, however, induced depotentiation in the presence of DCS and reversed TS-induced increase in surface GluR1. Together, this study has two important findings: (1) whether a memory trace remains intact or is erased depends on the interval between conditioning and extinction training and (2) DCS facilitates the reversal of memory trace. DCS-induced augmentation of extinction and reversal of GluR1 surface expression are likely mediated by DCS-facilitated endocytosis of AMPA receptors.

Effects of intra-amygdala infusion of CB1 receptor agonists on the reconsolidation of fear-potentiated startle.

The cannabinoid CB1 receptor has been shown to be critically involved in the extinction of fear memory. Systemic injection of a CB1 receptor antagonist prior to extinction training blocked extinction. Conversely, administration of the cannabinoid uptake inhibitor AM404 facilitated extinction in a dose-dependent manner. Here we show that bilateral infusion of CB1 receptor agonists into the amygdala after memory reactivation blocked reconsolidation of fear memory measured with fear-potentiated startle. The effect was dose-dependent and could be blocked by AM251, a specific CB1 receptor antagonist. In contrast, the effect of CB1 agonists on reconsolidation was no longer seen if memory reactivation was omitted. Concomitant with block of reconsolidation, CB1 agonist-treated animals did not exhibit shock-induced reinstatement or spontaneous recovery of fear. The absence of recovery was not attributable to permanent damage to the amygdala in WIN-treated rats, nor did the effect result from alteration of baseline startle or shock reactivity. These results suggest that CB1 agonists could impair fear memory via blocking reconsolidation.

Synaptic expression of glutamate receptor after encoding of fear memory in the rat amygdala.

Fear conditioning has been ascribed to presynaptic mechanisms, particularly presynaptic facilitation of transmission at thalamo- and cortico-amygdala synapses. Here, by labeling surface receptors with biotin or using membrane fractionation approaches, we report that fear conditioning resulted in an increase in surface expression of GluR1 subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the amygdala, whereas total GluR1 mRNA and protein levels were unchanged. The control group that received conditioned stimulus (CS) and unconditioned stimulus in an unpaired fashion did not present any increase, indicating that GluR1 increase was specific to the learning component of the task. Conditioning-induced increase in surface expression of GluR1 depended on the activation of N-methyl-d-aspartate receptors and protein kinases and required the synthesis of new proteins. CS-alone trials applied 24 h before training attenuated fear-potentiated startle and prevented conditioning-induced increase in surface expression of GluR1. Increase in GluR1 was also observed in the amygdala slices after delivery of tetanic stimulation that elicited long-term potentiation of synaptic transmission. Proteasome inhibitor increased surface expression of GluR1 in a time- and dose-dependent manner. Furthermore, pretraining administration of proteasome inhibitor into the amygdala facilitated the fear-potentiated startle. These results suggest that long-term memory formation is correlated with the change in synaptic expression of GluR1, and trafficking of GluR1 to the synaptic sites contributes at least in part to the expression of fear memory.