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

Abstract

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.

Figure 1. Recovery of fear after extinction training depends on the number of CS-alone trials.

(A) Plot of percent startle potentiation in context control and extinction rats. Rats received 10 light-shock pairings and were randomly assigned to 2 to 8 sessions of extinction training groups. Rats in 2 or 8 sessions of extinction groups received 2 or 8 sessions of 15 presentations of light-alone trials without footshock and memory retention was assessed 24 h later (Test 1). Context control rats were returned to the startle box at the equivalent time without receiving light-alone trials. All groups were also tested on day 8 (Test 2) and Day 15 (Test 3). ***p<0.001, **p<0.01, *p<0.05 vs. context controls. (B) Inhibition of spontaneous recovery by prolonged extinction training is blocked by mGluR5 antagonist. MTEP was administered intraperitoneally (2.5, 5 or 10 mg/kg) 60 min before light-alone trials or was infused into the amygdala (10 µg/per side) abilaterally 30 min before CS-alone trials. ***p<0.001, *p<0.05 vs. saline. (C) Distribution of cannula tips in the amygdala from rats infused with MTEP (10 µg/per side) in experiments B. (D) MTEP was without effect on the 30 CS-alone trials-induced extinction memory. (E) Distribution of cannula tips in the amygdala from rats infused with MTEP (10 µg/per side) in experiments D. (F) CPCCOEt (5 or 10 mg/kg) injected intraperitoneally 60 min before light-alone trials failed to affect 90 CS-induced extinction of fear memory.

Figure 2. Protein synthesis is required for 90 CS-alone trials-induced inhibition of spontaneous recovery.

(A) Rats were conditioned with 10 light-shock pairings and 24 h later received 6 sessions of 15 presentations of light-alone trials. Anisomycin was administered intraperitoneally (100 mg/kg, n = 7) 60 min before light-alone trials or was infused into the amygdala (62.5 µg/side, n = 7) 30 min before light-alone trials. ***p<0.001 vs. vehicle. (B) Distribution of cannula tips in the amygdala from rats infused with Anisomycin (62.5 µg/side, n = 7).

Figure 3. Effects of mGluR5 antagonist and protein synthesis inhibitor on prolonged extinction training-induced inhibition of spontaneous recovery in mice.

(A) On day 1, mice were placed in a context for 3 min and received a footshock every 60 sec for 5 times. On day 2, mice received intraperitoneal injection of saline, 2.5 mg/kg or 5 mg/kg of MTEP and 60 min later they were placed in the same context for 10 min without receiving footshock (extinction training). The extinction training was repeated 3 times with an interval of 30 min. Contextual freezing responses were measured as an index for memory retention on day 3 and day 8. *p<0.05 vs. saline or MTEP 2.5 mg. (B) CPCCOEt (10 mg/kg) injected intraperitoneally 60 min before extinction training failed to affect 90 CS-induced extinction of fear memory. (C) Same experimental procedure as (A) except anisomycin (62.5 µg/side, n = 10) or vehicle (n = 10) was infused into the hippocampus bilaterally 30 min before extinction training. *p<0.05 vs. vehicle. (D) Distribution of cannula tips in the hippocampus from mice infused with Anisomycin (62.5 µg/side, n = 10) or vehicle (n = 10).

Figure 4. Contextual fear conditioning and extinction in FMR1−/− mice and WT littermates.

(A) Acquisition of contextual fear conditioning. Mice were placed in a context for 3 min and received a foot shock every 60 sec for 5 times. Mice significantly increase freezing responses when compared to baseline (BL) measures before US presentation **p<0.01; ***p<0.001 vs. baseline (pairwise comparison test). (B) Extinction training. Mice underwent 3 extinction sessions with and interval of 30 min on day 2. *p<0.05; **p<0.01; ***p<0.001 (genotype effect). (C) Retention test. Freezing behavior was measured on day 3 and day 8 to evaluate long-term extinction memory *p<0.05; **p<0.01 (genotype effect). Data are expressed as mean ±s.e.m.

Figure 5. Reversal of conditioning-induced increases in surface expression of GluR1 and GluR2 is mediated by mGluR5.

(A) Representative blots and mean ± SEM of GluR1 (left) and GluR2 (right) immunoreactivities from rats that had been conditioned with 10 light-shock pairings and 24 h later received 2–6 sessions of 15 presentations of light-alone trials. Lateral (LA) and basolateral (BLA) amygdala tissues were dissected out, and surface GluR1 and GluR2 levels were determined by biotin labeling. **p<0.01, *p<0.05 vs. context control. (B) GluR1 and GluR2 surface levels were normalized to total protein in the left, and GluR1 and GluR2 intracellular levels were normalized to total protein in the right. Conditioning-induced increase in GluR1 and GluR2 surface levels and decrease in GluR1 and GluR2 intracellular levels were reversed after 90 CS-alone trials (CS). The effect of 90 CS-alone trials was blocked in a dose dependent manner by MTEP (2.5–10 mg/kg, i.p.) or by intra-amygdala infusion of MTEP (10 µg/side). ***p<0.001 vs. unpaired and naive, ### p<0.001, #p<0.05 vs. saline. (A) Representative immunoblots shown in B.

Figure 6. Effects of 90 CS-alone trials on conditioning-induced increases in AMPA/NMDA ratio and the expression of PSD-95 and SAP97.

(A) Plot of AMPA/NMDA ratios in naïve, unpaired, paired, 30 CS, 90 CS and 90 CS+MTEP rats. ***p<0.001 vs. paired and 30 CS, ###p<0.001 vs. 90 CS. Scale: 50 ms, 100 pA. (B) Representative blots and mean ± SEM of PSD-95 immunoreactivity from rats that have been conditioned and 24 h later received 2–6 sessions of 15 presentations of light-alone trials. Lateral (LA) and basolateral (BLA) amygdala tissues were dissected out, and PSD-95 levels were determined by Western blot analysis. ***p<0.001, **p<0.01 vs. context control. (C and D) Representative blots and mean ± SEM of PSD-95 and SAP97 immunoreactivities from rats that received 90 CS-alone trials. LA and BLA tissues were dissected out at various time points after extinction training as indicated, and PSD-95 and SAP97 levels were determined by Western blot analysis. *p<0.05, **p<0.01, ***p<0.001 vs. context control.

Figure 7. Tat-GluR2_3Y blocks the effect of 90 CS-alone trials on extinction.

(A) Rats received Tat-GluR2_3Y (15 pmol in 0.8 ml saline per side) or Tat- GluR2_3A (15 pmol in 0.8 ml saline per side) bilaterally into the amygdala 30 min before extinction training. Retention of memory was assessed 1 (Test 1), 6 (Day 8, Test 2) and 13 days (Day 15, Test 3) after extinction training. **p<0.01, ***p<0.001 vs. GluR2_3Y. (B) Distribution of cannula tips in the amygdala from rats infused with Tat-GluR2_3Y (△) or Tat- GluR2_3A (▴).