Activating mGlu3 Metabotropic Glutamate Receptors Rescues Schizophrenia-like Cognitive Deficits Through Metaplastic Adaptations Within the Hippocampus

Abstract

Background: Polymorphisms in GRM3, the gene encoding the mGlu₃ metabotropic glutamate receptor, are associated with impaired cognition and neuropsychiatric disorders such as schizophrenia. Limited availability of selective genetic and molecular tools has hindered progress in developing a clear understanding of the mechanisms through which mGlu₃ receptors regulate synaptic plasticity and cognition.

Methods: We examined associative learning in mice with trace fear conditioning, a hippocampal-dependent learning task disrupted in patients with schizophrenia. Underlying cellular mechanisms were assessed using ex vivo hippocampal slice preparations with selective pharmacological tools and selective genetic deletion of mGlu₃ receptor expression in specific neuronal subpopulations.

Results: mGlu₃ receptor activation enhanced trace fear conditioning and reversed deficits induced by subchronic phencyclidine. Mechanistic studies revealed that mGlu₃ receptor activation induced metaplastic changes, biasing afferent stimulation to induce long-term potentiation through an mGlu₅ receptor-dependent, endocannabinoid-mediated, disinhibitory mechanism. Selective genetic deletion of either mGlu₃ or mGlu₅ from hippocampal pyramidal cells eliminated effects of mGlu₃ activation, revealing a novel mechanism by which mGlu₃ and mGlu₅ interact to enhance cognitive function.

Conclusions: These data demonstrate that activation of mGlu₃ receptors in hippocampal pyramidal cells enhances hippocampal-dependent cognition in control and impaired mice by inducing a novel form of metaplasticity to regulate circuit function, providing a clear mechanism through which genetic variation in GRM3 can contribute to cognitive deficits. Developing approaches to positively modulate mGlu₃ receptor function represents an encouraging new avenue for treating cognitive disruption in schizophrenia and other psychiatric diseases.

Keywords: Cognition; Hippocampus; Schizophrenia; Synaptic plasticity; mGlu(3); mGlu(5).

Figure 1.. Activation of mGlu₃ receptors enhances associative learning and rescues schizophrenia-like cognitive deficits.

(A) Behavioral schematic. Mice received 3 pairings of a tone and mild foot-shock (0.5 mA), each separated by a 30-second trace period. Freezing time was quantified across the 3 traces. (B) Administration of the mGlu_2/3 agonist LY379268 (3 mg/kg, i.p., blue bars) 30 minutes prior to conditioning increased freezing at trace 2 and trace 3 relative to vehicle treated mice (grey bars) (**p<0.01, ****p<0.0001). Mice were treated with the mGlu₂ negative allosteric modulator (NAM) VU6001966 (10 mg/kg, i.p., red bars) or the mGlu₃ NAM VU0650786 (30 mg/kg, i.p., green bars) to isolate the effects of mGlu₂ and mGlu₃ receptor activation. LY379268 enhanced trace conditioning in VU6001966-treated mice (red bars, **p<0.01, ****p<0.0001 compared to vehicle (gray bars)), whereas the effect was blocked by the mGlu₃ NAM VU0650786 (##p<0.01 compared to LY379268, F_(3,72)=12.73 by two-way repeated measures ANOVA with Tukey’s post-hoc test, N=17-24 mice). (C) Schematic representing subchronic phencyclidine (scPCP) treatment regimen. After 7 days of habituation, mice were injected with scPCP (10 mg/kg) for 7 days. Trace fear conditioning experiments were performed 7 days after last PCP injection. (D) Behavioral schematic. Mice received 4 pairings of a tone, trace, and strong foot-shock (0.7 mA). (E) scPCP-treated mice (orange bars) froze less than vehicle controls (*p<0.05 compared to vehicle/vehicle; gray bars). Acute LY379268 treatment (30 minutes prior to the experiment; green bars) rescued scPCP-induced deficits in freezing (##p<0.01 compared to PCP/vehicle, red bars, F_(3,35)=8.027 by two-way repeated measures ANOVA with Tukey’s post-hoc test, N=8-13). Data are presented as mean ± SEM.

Figure 2.. mGlu₃ receptor activation enhances hippocampal long-term potentiation (LTP) through concerted signaling with mGlu₅ receptors.

(A) Schematic representing scPCP treatment regimen. After 7 days of habituation, mice were injected with scPCP for 7 days. Electrophysiology experiments were performed 7 days after last PCP injection. (B) Field excitatory postsynaptic potentials (fEPSPs) were recorded in the stratum radiatum of CA1 after electrical stimulation of the Schaffer collateral. Time course showing moderate LTP after a single application of theta burst stimulation (TBS) in slices from vehicle-treated mice (grey bar, n=7 slices). Slices from mice treated with scPCP displayed impaired LTP (orange bar n=6). Bath application of the mGlu_2/3 agonist LY379268 (100 nM) rescued TBS-LTP in slices from scPCP-treated mice (blue bar n=6). (C) Summary of averaged fEPSP slope of last 5 minutes of recordings from panel B (*p<0.05, compared to Vehicle, F_(2,16)=5.11, one-way ANOVA with Tukey’s post-hoc test). Insets for (C) are representative fEPSP traces for the various experimental conditions from (B) measured during baseline (1) and 55 min post stimulation (2). Scale bars represent 1 mV and 100 ms for all traces. Traces for each experimental condition are placed over respective bar graphs. (D) In control mice, LY379268 application enhanced LTP in response to TBS (blue squares, n=10). (E) Co-application of mGlu₃ NAM VU0650786 (VU786; 10 μM) blocked the enhanced LTP induced by LY379268 (green triangles, n=11) and had no effect on its own at baseline. Blue line displays LY379268 data from panel D. (F) The mGlu₅ NAM MTEP (1 μM) blocked enhanced LTP when co-applied with LY379268 (magenta diamonds, n=8) and had no effect on its own at baseline. (G) Summary of averaged fEPSP slope of last 5 minutes of recordings from panels D-F (****p<0.0001 compared to control, ***p<0.001 compared to LY379268+control, F_(3,42)=10.11, one-way ANOVA with Tukey’s post-hoc test). Data are presented as mean ± SEM.

Figure 3.. mGlu₃ receptor activation induces hippocampal metaplasticity to promote LTP

(A) Application of the mGlu_1/5 agonist DHPG (50 μM) induced long-term depression (LTD) of fEPSP slope (red circles, n=6 slices), while the mGlu_2/3 agonist LY379268 (100 nM) had no acute or long-term effects (blue circles, n=5). (B) Co-application of LY379268 abrogated DHPG-induced LTD (pink triangles, n=5). (C) Summary of averaged fEPSP slope of last 5 minutes of recordings from panels A-B (*p<0.05, compared to LY379268 alone, F_(2,13)=4.087, one-way ANOVA with Tukey’s post-hoc test). (D) Paired-pulse (PP) 1-Hz stimulation for 15 minutes induces mGlu₅-dependent LTD in control slices (grey circles, n=6). Coincidental application of LY379268 (100 nM) changes PP 1-Hz LTD into a long-term enhancement of fEPSP slope (blue circles, n=6). (E) The mGlu₃ NAM VU0650786 (VU786; 10 μM) blocked the metaplastic effects of LY379268 application (green diamonds, n=6). (F) Summary of averaged fEPSP slope of last 5 minutes of recordings from panels D and E (***p<0.001, compared to Control, F_(2,13)=17.13, p<0.001, one-way ANOVA with Tukey’s post-hoc test). All experiments were performed in presence of mGlu₁ NAM, VU0469650 (10 μM). Data are expressed as mean ± SEM.

Figure 4.. mGlu₃ receptor signaling regulates multiple modes of LTP

(A) A high concentration of LY379268 (300 nM) and sustained 0.5 Hz stimulation enhanced fEPSP slope in acute hippocampal slices (dark blue squares, n=6 slices). A modest concentration of LY379268 (100 nM) did not affect fEPSP slope on its own (light blue circles, n=8). Insets for (A) are representative fEPSP traces for the various experimental conditions measured during baseline (1) and 55 min post stimulation (2). Scale bars represent 1 mV and 50 ms for all traces. (B) The mGlu₃ NAM VU0650786 (10 μM) blocked the fEPSP enhancement induced by LY379268 application (green triangles, n=5). Blue line displays LY379268 (300 nM) data from panel A. (C) The mGlu₅ NAM MTEP (1 μM) blocked LY379268-induced LTP (pink diamonds, n=5). (D) Summary of averaged fEPSP slope of last 5 minutes of recordings from panels A-C (**p<0.01, compared to Control, F_(3,20)=5.672, p<0.01 one-way ANOVA with Tukey’s post-hoc test). (E) Brief priming stimulation (2, 10-second, 10 Hz trains) followed by TBS (red squares, n=5) enhanced fEPSP slope compared to TBS alone (gray circles, n=5). (F) The mGlu₃ NAM VU0650786 (10 μM) perfused before, during, and after priming stimulation blocked the enhanced LTP (green triangles, n=4). Red lines display TBS with priming data from panel E. (G) The mGlu₅ NAM MTEP (1 μM) perfused throughout priming stimulation blocked the enhanced LTP (pink diamonds, n=4). (H) Summary of averaged fEPSP slope of last 5 minutes of recordings from E-G (**p<0.01, compared to Control, F_(3,14)=9.136, p<0.01 one-way ANOVA with Tukey’s post-hoc test). Data are presented as mean ± SEM.

Figure 5.. mGlu₃ receptor activation decreases evoked inhibitory transmission onto hippocampal pyramidal neurons.

(A) Electrically-evoked inhibitory postsynaptic currents (eIPSCs) were recorded from pyramidal cells in CA1. A high concentration of the mGlu_2/3 agonist LY379268 (300 nM) reduced the amplitude of eIPSCs (blue circles, n=6 cells). Insets for (A) are representative eIPSCs traces measured during baseline (1) and last 2 min of the recordings from the time course (2). Scale bars represent 200 pA and 50 ms. (B) Co-application of the mGlu₃ NAM, VU0650786 (20 μM) blocked the LY379268-induced decrease in eIPSCs (green squares, n=10). (C) The CB1 antagonist AM251 (2 μM) blocked the effects of LY379268 on eIPSC amplitude (red circles, n=6). (D) Summary of the last 2 min of the recordings from the time course experiments in panels A-C (*p<0.05, **p<0.01 compared to control, F_(2,19)= 8.042, one-way ANOVA with Tukey’s post-hoc test). (E) In field potential configuration, AM251 (2 μM) blocked LTP (red squares, n=5 slices) induced by high concentration of LY379268 (300 nM) (blue circles, n=5). (F) Summary of averaged fEPSP slope of last 5 minutes of recordings from panel E (*p<0.05, compared to Control, t₍₈₎=2.86, Student’s t-test). (G) AM251 (red squares, n=10) blocked the ability of LY379268 (100 nM) to enhance LTP following TBS (blue circles, n=5). (H) Summary of averaged fEPSPs slope of last 5 minutes of recordings from panel H. (**p<0.01, compared to Control, t₍₁₃₎=3.617, Student’s t-test). Data are expressed as mean ± SEM.

Figure 6.. Generation and characterization of conditional Grm3^Fl/Fl mice.

(A) Schematic of the procedure for generating the floxed Grm3 clones. (Top) Wild-type Grm3 locus surrounding exon 3. (Middle) Floxed Grm3 locus where LoxP sites are inserted flanking exon 3 of Grm3. (Bottom) Cre-mediated recombination leading to loss of Grm3 exon 3 and Frt site. (B) PCR from the DNA of mice homozygous for the WT allele (277 bp) and mice heterozygous and homozygous for the floxed allele (413 bp). (C) CMV-Cre mediated recombination in Grm3^Fl/Fl mice excises Grm3 exon 3, leading to loss of WT allele (~1900 bp) and generation of a smaller recombination allele (671 bp). (D) RT-PCR from the hippocampi of WT and Grm3-CMV KO mice. (E) Western blot depicting loss of mGlu₃ protein from the hippocampus of Grm3-CMV KO mice (red bar) relative to Grm3^Fl/Fl controls (blue bar). (F) Bar graph depicting quantification of Western blots. Data are presented as mean ± SEM, N=3-6 mice. (***p<0.001 compared to Grm3^Fl/Fl mice, t₍₇₎= 7.018, Student’s t-test). (G) Confocal 40X RNAscope in situ hybridization images showing loss of Grm3 mRNA (red) from both neurons (Syn2; Synapsin, green) and astrocytes (Slc1a3; GLAST, magenta) in Grm3-CMV KO mice. Scale bar = 20 μm for the merged left panel image, and 10 μm for the 3X images.

Figure 7.. Grm3-CamKII KO mice display selective ablation of mGlu₃ receptor transcript in pyramidal cells of the hippocampus.

(A) Characterization of Grm3-CamKII KO mice. Merged confocal 20X tile scan image of a coronal section highlighting decreased Grm3 mRNA expression in the CA1 of 7-8-week-old Grm3-CaMKII KO mice. Scale bars denote 500 μm. (B) Representative confocal 40X RNAscope in situ hybridization images showing loss of Grm3 mRNA (green) from pyramidal neurons (Slc17a7; vGluT1, red) of CA1, while Grm3 mRNA expression is intact in astrocytes (Slc1a3; GLAST, gray). Scale bars denote 20 μm for the left panel image and 10 μm for the right panels (per genotype: Grm3^Fl/Fl=3; Grm3-CaMKII KO=4). (C) The percentage of Slc17a7-positive cells (vGluT1) with Grm3 mRNA was decreased in Grm3-CaMKII KO mice (red bar, n/N = 16/4 slices/mice) as compared to WT controls (blue bar, n/N = 15/3) (****p<0.0001 compared to Grm3^Fl/Fl mice, t₍₂₉₎ = 19.40, Student’s t-test). (D) The percentage of Slc1a3-positive cells (GLAST) with Grm3 mRNA was not different between Grm3-CaMKII KO mice (red bar, n/N = 14/4) and controls (blue bar, n/N = 9/3) (t₍₂₁₎ = 0.7951, Student’s t-test, p=0.4355).

Figure 8.. Neuronal mGlu receptors mediate cognitive enhancement following mGlu₃ receptor activation

(A) LY379268 (100 nM) failed to enhance LTP after TBS in slices from Cre+ knockout mice (blue circles, n=6) relative to Cre− littermate controls (grey circles, n=5). (B) Summary of averaged fEPSPs of last 5 minutes of recordings from panel C (*p<0.05 compared to Cre−, t₍₉₎= 2.506, Student’s t-test). (C) Systemic treatment with LY379268 (3 mg/kg) enhances the expression of trace fear conditioning in Cre− littermate controls (hatched grey bars, N=9 mice) but not in Cre+ knockout mice (hatched blue bars, N=7 mice) as compared to treatment with vehicle (gray bars, Cre−, N=7 mice; blue bars, Cre+, N=7 mice) (***p<0.001 compared to Cre− Vehicle, F_(3,104)=60.24, two-way repeated measures ANOVA with Tukey’s post-hoc test). (D) LY379268 (100 nM) potentiates LTP after TBS stimulation in Cre− controls (grey circles, n=5) but not Cre+ slices (red circles, n=5). (E) Summary of averaged fEPSPs of last 5 minutes of recordings (*p<0.05 compared to Cre−, t₍₈₎= 2.7, Student’s t-test). (F) Loss of enhanced freezing expression following LY379268 treatment (hatched bars) in Cre+ mice (red hatched bars, 7 mice) as compared to Cre− littermate controls (grey hatched bars, 8 mice) (**p<0.01 compared to Cre− Vehicle, F_(3,72)=2.788, two-way repeated measures ANOVA with Tukey’s post-hoc test, N=4-9 mice). Data are expressed as mean ± SEM.