Facilitation of extinction learning for contextual fear memory by PEPA: a potentiator of AMPA receptors

Abstract

Contextual fear memory is attenuated by the re-exposure of mice to the context without aversive stimulus. This phenomenon is called extinction. Here, we report that a potentiator of AMPA receptors, 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluorophenoxyacetamide (PEPA), potently facilitates extinction learning in mice. C57BL/6J mice were exposed to novel context and stimulated by electrical footshock. After 24 h (extinction training) and 72 h (extinction test), the mice were repeatedly exposed to the context without footshock and the duration of their freezing response was measured. The duration of freezing response in the extinction test was consistently shorter than the value in extinction training. Intraperitoneal injection of PEPA 15 min before extinction training remarkably reduced the duration of freezing responses during the extinction training and test, compared with the vehicle-injected control mice. This action of PEPA on extinction was dose-dependent and inhibited by NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide), an AMPA receptor antagonist. PEPA had no effect on acquisition and consolidation of fear memory itself. Electrophysiological studies suggested that PEPA activates the neural network much more potently in the medial prefrontal cortex (mPFC) than in the basolateral amygdala and hippocampal CA1 field. Quantitative PCR studies suggested the pronounced expression of PEPA-preferring AMPA receptor subunits (GluR3 and GluR4) and a splice variant (flop) in the mPFC. An intra-mPFC injection of PEPA facilitated the extinction much more potently than an intra-amygdala injection of PEPA did. These results suggest that PEPA facilitates extinction learning through AMPA receptor activation mainly in the mPFC.

Figure 1.

PEPA facilitates extinction learning. A, Experimental design for B–D. Mice were trained for the contextual fear conditioning and tested 24 h (extinction training) and 72 h (extinction test) later. PEPA or vehicle (V) was injected (i.p.) 15 min before extinction training. B, C, Dose-dependent reduction of freezing time during extinction training (B) and extinction test (C). Data represent mean + SEM (n = 9–11 for each group). D, Reduction in freezing time between the extinction training and extinction test based on the data in B and C. E, Experimental design for F. Mice were trained for the contextual fear conditioning and tested 72 h later (test) without extinction training. PEPA or vehicle were injected 24 h after conditioning. F, PEPA did not reduce freezing time in the test when extinction training was skipped (n = 10 each). *Statistically significant (p = 0.002 vs V in B; p < 0.001 vs V in C, and p = 0.005 vs V in D, Bonferroni/Dunn test). G, H, Changes in freezing time during the 360 s extinction training (G) and test (H) in groups injected with PEPA (30 mg/kg, n = 11) or vehicle (n = 9). Data used in B and C were reanalyzed for the time course of changes in freezing time (bin = 60 s). *Statistically significant (p = 0.002, 0.002, and 0.001 for bins 3–5, respectively, vs V in G, and p < 0.02 for all vs V in H, two-tailed Student's t test).

Figure 2.

The effect of PEPA on extinction learning is mediated by AMPA receptors. A, Experimental design. Mice were injected with saline (SA) or NBQX (NB, 1 mg/kg, s.c.) 60 min before vehicle (V) or PEPA (P; 30 mg/kg) injection (i.p.) and extinction training and testing were performed. B, NBQX antagonizes the effect of PEPA on extinction learning. *Statistically significant (p < 0.001 and p = 0.002 vs SA/V in extinction training and test, respectively, Bonferroni/Dunn test; ^#p = 0.004 and 0.001 vs SA/P in extinction training and test, respectively, Bonferroni/Dunn test). Error bars indicate + SEM.

Figure 3.

PEPA does not influence locomotion and anxiolytic drug-sensitive behavior. Mice were injected (i.p.) with vehicle (V) or PEPA (P; 30 mg/kg) 15 min before the open-field test and hole-board test. A, B, C, Locomotion assessed by total distance that mice traveled for 5 min (A), the percentage of time that the mice spent in the perimeter of the open-field (B), and the number of head-dipping behaviors in the hole-board test (C). None of the values differed significantly between the groups injected with vehicle (n = 6 for all tests) or PEPA (n = 7 for all tests). Error bars indicate + SEM.

Figure 4.

PEPA more potently activates the neural circuit in the mPFC than in the BLA or hippocampal CA1 field. A, mPFC-type I, An example of type I modulation by PEPA of synaptic currents recorded from layer V pyramidal cells in response to electrical stimulation of the mPFC layer II in mouse brain slices. A, mPFC-type II, An example of type II modulation by PEPA of similar synaptic currents. The arrow indicates epileptiform activity. C indicates CNQX. B, Dose-dependent augmentation of synaptic currents by PEPA in cells showing type I modulation. C, BLA-NM, An example of synaptic currents that were not affected by PEPA. NM, no modulation. The synaptic currents were recorded from a BLA pyramidal cell in response to electrical stimulation of the external capsule in mouse brain slices. BLA-type I, An example of type I modulation by PEPA of similar synaptic currents. D, CA1-NM, An example of synaptic currents that were not affected by PEPA. The synaptic currents were recorded from a neuron in the CA1 pyramidal layer in response to electrical stimulation of stratum radiatum in mouse hippocampal slices. CA1-type I, An example of type I modulation by PEPA of similar synaptic currents. NM-CYZ, An example of similar synaptic currents that were not affected by PEPA but potently enhanced by CYZ. Calibrations: 20 ms, 100 pA. E, Summary of the action of PEPA (50 μm) in the mPFC, BLA, and CA1-field (the total number of cells tested is shown in parentheses). The proportion of the cells exhibiting NM, type I, and type II modulations is indicated by color in each bar. Error bars indicate + SEM.

Figure 5.

Quantitative PCR analysis of the expression of AMPA receptor mRNA. A, Expression levels of AMPA receptor subunit (GluR1–4) mRNAs in the mPFC, amygdala (AMY), and hippocampus (HIPP). The values were normalized to the value obtained for β-actin mRNA. n = 4 for mPFC and HIPP, and = 3 for AMY. *^,#Statistically significant (*p = 0.017 vs AMY and ^#p < 0.001 vs HIPP in R3, ^#p < 0.001 vs HIPP in R4, two-tailed Student's t test). B, Percentage of flop variant mRNAs in GluR1–4 subunits in the mPFC and amygdala. n = 8 and 7 for mPFC and AMY, respectively. *Statistically significant (p < 0.001, p = 0.006, p = 0.009, and p = 0.012 vs AMY in R1, R2, R3, and R4, respectively). Error bars indicate + SEM.

Figure 6.

Intra-mPFC injection of PEPA (0.02 μg) 30 min before extinction training facilitates extinction learning. A, Experimental design. Mice were trained for the contextual fear conditioning and tested 24 h (extinction training) and 72 h (extinction test) later. PEPA or vehicle (V) were microinjected into the mPFC or amygdala 30 min before extinction training. B, Location of the injection cannulas within the mPFC. Open circles, Vehicle; shaded circles, PEPA. C, Freezing time of mice microinjected with vehicle (n = 6) or PEPA (n = 5) into the mPFC in extinction training and test. *Statistically significant (p = 0.004 and 0.029 vs V in extinction training and test, respectively, two-tailed Student's t test). D, E, Changes in freezing time during the 360 s extinction training (D) and test (E) in groups injected with vehicle or PEPA. Data used in C were reanalyzed for the time course of changes in freezing time (bin = 60 s). *Statistically significant (p = 0.004, 0.017, 0.027, and 0.014 for bins 2–5, respectively, vs V in D, and p < 0.04 for all vs V in H, two-tailed Student's t test). F, Location of the injection cannulas within the amygdala. Open circles, vehicle; shaded circles, PEPA. G, Freezing time of mice microinjected with vehicle (n = 6) or PEPA (n = 5) into the amygdala in extinction training and test. The values in vehicle- and PEPA-injected groups are not significantly different in either extinction training or extinction test (p = 0.120 and 0.250 in extinction training and test, respectively, two-tailed Student's t test). Error bars indicate + SEM.