Contextual Fear Extinction Induces Hippocampal Metaplasticity Mediated by Metabotropic Glutamate Receptor 5

Abstract

Dysregulated fear memory can lead to a broad spectrum of anxiety disorders. The brain systems underlying fear memory are manifold, with the hippocampus being prominently involved by housing fear-related spatial memories as engrams, which are created and stored through neural changes such as synaptic plasticity. Although metabotropic glutamate (mGlu) receptors contribute significantly to both fear behavior and hippocampal synaptic plasticity, the relationship between these two phenomena has not been fully elucidated. Here, we report that contextual fear extinction induces a novel form of metaplasticity mediated by mGlu5 at the hippocampal SC-CA1 synapse. Further, blockade of mGlu5 prevents both contextual fear extinction and expression of this metaplasticity. This form of metaplasticity was absent in a mouse model of MECP2-duplication syndrome, corresponding to a complete deficit in extinction learning. These findings suggest that mGlu5-dependent metaplasticity within the hippocampus may play a critical role in extinction of contextual fear.

Figure 1.

mGlu receptor-dependent LTD is converted to LTP in the hippocampus after fear conditioning. (A) Schematic of fear conditioning procedure. (B) Graph representing the percent time freezing to the context on T1 (***P < 0.001, compared with naïve, t₍₂₂₎ = 10.85, Student’s t test; naïve n = 12, Cond n = 12). (C) Input–output function measured at SC-CA1 synapse in hippocampal slices (black symbols, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with naïve; P < 0.0001, F_(1,14) = 8.77, two-way ANOVA; naïve n = 9, Cond + T1 n = 10). (D) Paired-pulse ratio (PPR) at SC-CA1 synapse (P = 0.884, two-way ANOVA, naïve n = 9, Cond + T1 n = 10). (E) Time course showing the effects of PP-LFS on fEPSP slope in acute slices of naïve (black symbols, n = 9) and Cond + T1 (gray symbols, n = 10) mice. Representative traces (1) and (2) correspond to baseline (1) and 60 min post PP-LFS (2). (F) Averaged fEPSPs from last 5 min of a 60 min recording show significant difference in naïve compared with conditioned groups (**P < 0.01, compared with naïve, t₍₁₂₎ = 4.16, Student’s t test). Data are expressed as mean ± standard error of the mean (SEM).

Figure 2.

Fear conditioning-induced metaplasticity is dependent on mGlu₅, NMDA and CB1 receptors. (A) Time course showing the effects of PP-LFS on fEPSP slope in acute slices from Cond + T1 mice with MTEP (1 μM) perfusion throughout experiment (n = 5). (B) Time course showing the effects of PP-LFS on fEPSP slope in acute slices with VU0649650 (10 μM) perfused throughout the entire experiment (n = 6). (C) Time course showing the effects of PP-LFS on fEPSP slope in acute slices with AP5 (50 μM) perfused throughout entire experiment (n = 8). (D) Time course showing the effects of PP-LFS on fEPSP slope in acute slices with AM251 (2 μM) perfused throughout entire experiment (n = 5). Summary of averaged fEPSPs from Figures 1F and 2A–D. (**P < 0.01 compared with naïve/vehicle; P = 0.0005, F_(5,37) = 5.75, one-way ANOVA). Data are expressed as mean ± SEM.

Figure 3.

Fear extinction mediates the shift to mGlu receptor-dependent LTP in the hippocampus. (Ai) Schematic of behavioral paradigm for mice that underwent conditioning without re-exposure to the context (Cond only). (Aii) Schematic of behavioral paradigm for mice that underwent conditioning (Cond) followed by contextual fear extinction (T1–T5). (B) Graph of percent time spent freezing over 5 contextual extinction sessions (*P < 0.05, **P < 0.01, ***P < 0.001 compared with T1; P = 0.0002, F_(4,75) = 6.37, one-way repeated measures ANOVA, n = 16). (C) Input–output function of fEPSPs in acute slices (Extinction: gray symbols, *P < 0.05, **P < 0.01, ***P < 0.001 compared with Cond only: white symbols; P < 0.0001, F_(1,9) = 10.08, two-way ANOVA with repeated measure, Cond only n = 6, extinction n = 7). (D) Time course showing the effects of PP-LFS on fEPSP slope in acute slices from Cond only (white symbols, n = 6) and extinction (gray symbols, n = 7) groups. (E) Averaged fEPSPs from last 5 min of recording (gray bar in (D)) show significant difference in slices from mice that underwent extinction compared with conditioned only (***P < 0.001, t₍₁₃₎ = 5.63, Student’s t test). Data are expressed as mean ± SEM.

Figure 4.

Contextual fear extinction and hippocampal metaplasticity are prevented by mGlu₅ antagonism. (A) Schematic of behavioral paradigm. (B) Fear extinction was significantly impaired by MTEP treatment (black symbols, 5 mg/kg, i.p.; 30 min prior to each trial) compared with vehicle-treated mice (gray symbols) (***P < 0.001 compared with vehicle; P < 0.0001, F_(1,29) = 29.68, two-way repeated measures ANOVA, vehicle n = 16, MTEP n = 15). (C) Input–output function in acute slices of vehicle-Ext compared with MTEP-Ext mice (gray vs. black symbols, respectively, *P < 0.05, **P < 0.01, ****P < 0.0001 compared with MTEP treated; P < 0.0001, F_(1,9) = 9.93, two-way repeated measures ANOVA, vehicle-Ext n = 4, MTEP-Ext n = 7). (D) Time course showing the effects of PP-LFS on fEPSP slope in acute slices of MTEP-Ext mice (black symbols, n = 4) and vehicle-Ext slices (gray symbols, n = 7). (E) Averaged fEPSPs from last 5 min of a 60 min recording show significant difference in vehicle-Ext compared with MTEP-Ext (**P < 0.01 compared with vehicle-Ext; P < 0.01, t₍₁₁₎ = 4.23, Student’s t test). Data are expressed as mean ± SEM.

Figure 5.

Contextual fear extinction and mGlu receptor-dependent LTP is restored following termination of MTEP treatment. (A) Schematic of behavioral paradigm. (B) Graphical representation of percentage of time freezing during extinction (T1–T5); MTEP (black symbols, T1–T5), vehicle (gray symbols, T6–T10) (*P < 0.05, **P < 0.01, ***P < 0.001 compared with T5; P < 0.0001, F₍₉₁₁₀₎ = 10.53, one-way repeated measures ANOVA, n = 12). (C) Time course showing the effects of PP-LFS on fEPSP slope in acute slices from mice that underwent 10 total days of extinction (MTEP, T1–T5; Vehicle, T6–T10), which resulted in LTP (n = 6). Data are expressed as mean ± SEM.

Figure 6.

Temporally mediated spontaneous recovery of contextual fear corresponds to a loss of mGlu receptor-dependent LTP. (A) Schematic of behavioral paradigm. (B) Graphical representation of percentage of time freezing during extinction (T1–T5) and spontaneous recovery test (SR, 30 days post T5). (*P < 0.05, **P < 0.01, ***P < 0.001 compared with T1, ^##P < 0.01 compared with T5; P < 0.0001, F_(5,11) = 8.01, one-way ANOVA repeated measures, n = 12). (C) Input–output function of fEPSPs in acute slices after a 30-day delay (black symbols, n = 7) compared with 1 day after T5 (gray symbols, n = 7) (**P < 0.01, ***P < 0.001, ****P < 0.0001 compared with 30-day delay; P < 0.0001, F_(1,9) = 8.11, two-way ANOVA repeated measures). (D) Time course showing the effects of PP-LFS on fEPSP slope in acute slices from mice that underwent a 30-day delay (black symbols, n = 7) compared with one day after T5 (gray symbols, n = 7). Data are expressed as mean ± SEM.

Figure 7.

Contextual fear extinction recall is disrupted by mGlu₅ antagonism. (A) Schematic of behavioral paradigm. (B) Graphical representation of percentage of time freezing during extinction after systemic administration of vehicle (gray symbols, T1–T5) or MTEP (5 mg/kg i.p., black symbols, T6) (*P < 0.05, **P < 0.01, ***P < 0.001 compared with T1, ^###P < 0.001 compared with T5; P < 0.0001, F_(5,10) = 8.38, one-way ANOVA repeated measures, n = 11). (C) Diagram of injector tip placement for all mice that received hippocampal CA1 infusions. (D) Graphical representation of percentage of time freezing during extinction after direct infusion of vehicle (gray symbols, T1–T5) or MTEP (1 μg/side, black symbols, T6) (**P < 0.01, ***P < 0.001 compared with T1, ^#P < 0.05 compared with T5; F_(5,9) = 8.32, one-way ANOVA repeated measures, n = 10). Data are expressed as mean ± SEM.

Figure 8.

MECP2 Duplication Syndrome model mice display impairments in contextual fear extinction and mGlu₅-dependent metaplasticity. (A) Schematic of behavioral paradigm. (B) Graphical representation of percentage of time freezing during extinction in WT littermate mice (black symbols) and MeCP2-Tg1 mice (gray symbols) (*P < 0.05, **P < 0.01, ***P < 0.001 compared with WT; P < 0.001, F_(1,15) = 16.98, two-way ANOVA with repeated measures, WT n = 9, MeCP2-Tg1 n = 8). (C) Time course showing the effects of PP-LFS on fEPSP slope in acute slices from WT mice (black symbols, n = 6) and MeCP2-Tg1 mice (gray symbols, n = 9). Representative traces (1) and (2) correspond to baseline (1) and 60 min post PP-LFS (2). (D) Averaged fEPSPs from the last 5 min of a 60 min recording (gray bar in (C)) (***P < 0.001 compared with WT; P < 0.001, t₍₁₃₎ = 4.97 Student’s t-test). Data are expressed as mean ± SEM.