Metabotropic glutamate receptor 5/Homer interactions underlie stress effects on fear

Abstract

Background: Glutamatergic transmission is one of the main components of the stress response; nevertheless, its role in the emotional stress sequelae is not known. Here, we investigated whether interactions between group I metabotropic glutamate receptors (metabotropic glutamate receptor 1 and metabotropic glutamate receptor 5 [mGluR5]) and Homer proteins mediate the delayed and persistent enhancement of fear induced by acute stress.

Methods: Antagonists and inverse agonists of metabotropic glutamate receptor 1 and mGluR5 were injected into the hippocampus after immobilization stress and before contextual fear conditioning. Metabotropic glutamate receptor 5 was displaced from constitutive Homer scaffolds by viral transfection of Homer1a or injection of Tat decoy peptides. The effects of these manipulations on stress-enhanced fear were determined.

Results: We show that stress induces interactions between hippocampal mGluR5 and Homer1a; causes a sustained, ligand-independent mGluR5 activity; and enhances contextual fear. Consistent with this mechanism, enhancement of fear was abolished by delayed poststress application of inverse agonists, but not antagonists, of mGluR5. The effect of stress was mimicked by virally transfected Homer1a or injection of Tat-metabotropic glutamate receptor C-tail decoy peptides into the hippocampus.

Conclusions: Constitutive activation of mGluR5 is identified as a principal hippocampal mechanism underlying the delayed stress effects on emotion and memory. Inverse agonists, but not antagonists, of mGluR5 are therefore proposed as a preventive treatment option for acute and posttraumatic stress disorders.

Figure 1

Stress-enhanced fear conditioning requires ongoing activity of mGluR5. (A) Design of the stress/conditioning phase of the experiment indicating the time window between stress and fear conditioning. (B) Fear conditioning performed 3 and 6 hrs after stress resulted in a significant enhancement of fear tested 24 hr later. (C) The enhancing effect of stress on fear lasted for at least a month. * P < 0.05, *** P < 0.001 vs non-stress/vehicle; ### P < 0.001 vs stress/vehicle. The lightning bolt sign indicates footshock. The number of mice per group is marked on the bars for experiment.

Figure 2

Stress-enhanced fear conditioning requires ligand-independent but not ligand-dependent activation of mGluR5 or mGluR1 in the hippocampus. (A) Intrahippocampal injection of 50 nmol/mouse MPEP up to 5 hr post-stress reduced enhancement of fear. (B) The potent mGluR5 inverse agonist MTEP injected intrahippocampally 5 hr after stress prevented stress enhancement contextual fear. (C) Intrahippocampal injection of the mGluR competitive antagonist MCPG 5 hr after stress did not prevent the enhancement of fear. (D) Intrahippocampal injection of the mGluR1 antagonist CPCCOet (100 nmol/mouse), or inverse agonist BAY36-7620 (100 nmol/mouse) 5 hr post-stress did not reverse stress-enhanced fear. ** P < 0.01, *** P < 0.001 vs non-stress/ vehicle; # P < 0.05, ## P < 0.01, ### P < 0.001 vs stress/vehicle.

Figure 3

mGluR5/Homer1a interactions are induced by stress. (A) A model of mGluR5/Homer interactions showing ligand-dependent and independent mGluR5 activity and displacement of mGluR5 from Homer scaffolds by Homer1a. This may indirectly alter the effect of Homer1-3 on calcium homeostasis mediated by ryanodine and inositol triphosphate channels. (B) A representative immunoblot showing hippocampal mGluR5/Homer1a and mGluR5/Homer1b/c interactions 3 and 6 hrs after stress. Left: coimmunoprecipitation (CoIP), Right: input; Input/IgG is the input sample used for IgG co-immunoprecipitation; N: naive. The co-immunoprecipitation experiment was replicated with 6 pull-downs of independent sample pools, each prepared from 2–5 individual membrane fractions/group (a total of N=12–15 hippocampi/group). (C) Quantification of the immunoblot signals showing mGluR5/Homer1a interactions significantly increased after stress whereas (D) mGluR5/Homer1b/c interactions exhibited a significant decrease. * P < 0.05, ** P < 0.01, *** P < 0.001 vs naïve.

Figure 4

Overexpression of Homer1a mimics the effects of stress. (A) Mice injected with rAAV-H1aV or and rAAV-GFP show robust protein expression in the dorsal hippocampus 10 days later. Homer1a antibody revealed strong signals at 10, but not 5 days after rAAV-H1aV injection. Both viral vectors showed high levels of expression as detected by GFP antibody. H1aV levels and distribution in CA1 dorsohippocampal neurons 5 and 10 days after viral injection. White arrows indicate Homer1a-positive neurons. (B) H1aV interacts with mGluR5 as revealed by robust co-immunoprecipitation of both proteins 10 days after injection of rAAV-H1aV, whereas mGluR5/Homer1b/c interactions decrease. Input/IgG is the input sample used for IgG co-immunoprecipitation; The co-immunoprecipitation experiment was replicated with 3 pull-downs of independent sample pools, each prepared from 2–4 individual membrane fractions/group (a total of N=10–13 hippocampi/group). (C) Quantification of the immunoblot signals showing significant increase of mGluR5/H1aV interaction when compared to all other groups. The data were normalized to H1aV because there was no detectable signal in the naïve control. (D) Quantification of the immunoblot signals showing significant decrease of mGluR5/Homer1b/c signals 10 days after hippocampal injection of rAAV-H1aV. (E) Overexpression of H1aV leads to enhanced contextual fear conditioning. Injection of MPEP 1 hr prior to training reverses this enhancement. * P < 0.05, *** P < 0.001 vs naïve, +++ P < 0.001 vs rAAV-GFP, ## P < 0.01 vs rAAVH1aV/Vehicle.

Figure 5

Uncoupling mGluR5 from long Homer isoforms mimics the effects of stress on fear. (A) A model of endogenous mGluR5/Homer interactions showing displacement of mGluR5 from Homer scaffolds by TAT decoy peptides. (B) Quantification of coimmunoprectpitation signals after intrahippocampal injection of active peptide causing reduction of mGluR5/Homer1b/c interactions at a dose of 500 but not 250 ng/mouse. The scramble peptide was ineffective. The co-immunoprecipitation experiment was replicated with 4 pull-downs of independent sample pools, each prepared from 2–3 individual membrane fractions/group (a total of N=10–12 hippocampi/group). (C) Intrahippocampal injection of active peptide at a dose of 500 ng/mouse 1 hr before training enhanced fear conditioning of non-stressed mice when compared to the scramble- or vehicle-injected controls. Prior stress prevented further enhancement of contextual fear by TAT peptides. * P < 0.05, ** P < 0.01 vs vehicle, # P < 0.05 vs Scramble.

Figure 6

TAT peptides reduce mGluR5 interactions and enhance fear in an mGluR5-dependent manner. (A) FITC-tagged active peptide, contrary to the FITC-tagged TAT_38–48 peptide, readily entered into cultured neurons (upper panels) and in CA1 pyramidal cells in vivo (lower panels). (B) A representative co-immunoprecipitation image showing reduced mGluR5/Homer1b/c interactions after injection of active TAT. Input/IgG is the input sample used for IgG co-immunoprecipitation. The co-immunoprecipitation experiment was replicated with 3 pull-downs of independent sample pools, each prepared from 2–3 individual membrane fractions/group (a total of N=6–9 hippocampi/group.) (C) Quantification of the immunoblot signals showing a significant effect of active TAT but not impermeable TAT_38–48 on mGluR5/Homer1b/c interactions. (D) Intrahippocampal injection of 500 ng/mouse of FITC-tagged active peptide 1hr before training enhanced fear when compared to the cell impermeable TAT_38–48 peptide or vehicle. (E) Enhancement of fear by active TAT peptide was completely abolished by MPEP (50 nmol/mouse) injected 15 min later. * P < 0.05, ** P < 0.01 vs vehicle, ++ P < 0.01 vs TAT_38–48, ## P < 0.01 vs Active + vehicle.