Input-specific regulation of glutamatergic synaptic transmission in the medial prefrontal cortex by mGlu2/mGlu4 receptor heterodimers

Abstract

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that regulate various aspects of central nervous system processing in normal physiology and in disease. They are thought to function as disulfide-linked homodimers, but studies have suggested that mGluRs can form functional heterodimers in cell lines. Using selective allosteric ligands, ex vivo brain slice electrophysiology, and optogenetic approaches, we found that two mGluR subtypes-mGluR2 and mGluR4 (or mGlu₂ and mGlu₄)-exist as functional heterodimers that regulate excitatory transmission in a synapse-specific manner within the rodent medial prefrontal cortex (mPFC). Activation of mGlu₂/mGlu₄ heterodimers inhibited glutamatergic signaling at thalamo-mPFC synapses but not at hippocampus-mPFC or amygdala-mPFC synapses. These findings raise the possibility that selectively targeting these heterodimers could be a therapeutic strategy for some neurologic and neuropsychiatric disorders involving specific brain circuits.

Fig. 1.. Effects of L-AP4 on optogenetically evoked glutamatergic transmission at thalamo-, hippocampo-, and amygdala-mPFC synapses.

(A to C) Schematic diagram showing the injection of AAV5-CaMKIIa-hChR2(E123T/T159C)-EYFP into the mediodorsal thalamic nucleus (MDT; A), ventral hippocampus (vHPC; B), or basolateral amygdala (BLA; C), each with a representative image of EYFP-tagged ChR2 expression in the injection site 3 to 5 weeks after viral injection (calibration bar, 200μm), and a second schematic (right) depicting the acquisition of ex vivo whole-cell recordings from mPFC layer V pyramidal cells in response to light stimulation of ChR2:EYFP expressed exons. Bottom panels show the effect of L-AP4 on both oEPSCs over time and paired-pulse ratios (PPR) at each synapse type. Data are mean ± SEM from N = 8 neurons from total of 7 mice (A), and 7 neurons from 5 (B) or 4 (C) mice. Synapse-specific oEPSCs versus baselines were compared by Wilcoxon matched-pairs tests: **P <0.01 (A), P > 0.1 (B), and P > 0.9 (C). PPRs were also compared by Wilcoxon matched-pairs tests: **P < 0.01 (A), P > 0.9 (B), and P > 0.2 (C). Abbreviations: dHPC = dorsal hippocampus, CTX = cortex, vCA1= ventral CA1, MB = midbrain, CP = caudal putamen. Calibration bars for oEPSC traces: 100pA/20ms (A), 50pA/20ms (B), and 20pA/20ms (C). The gray-shaded vertical bar in the time course indicates the time points used to be averaged for statistical comparisons to baseline. For reference but not marked in the figure is the statistical comparison of oEPSCs and PPR at 14-15 min after L-AP4 application at hippocampo-mPFC synapses (B): P < 0.05 and P > 0.9, respectively, by Wilcoxon matched-pairs test.

Fig. 2.. Effects of L-AP4 and mGlu₄ PAMs with different pharmacologic profiles on glutamatergic transmission at thalamo-mPFC synapse.

(A) Time courses (left) and dot plot (right) summarizing the effects of L-AP4 at different concentration on oEPSCs at thalamo-mPFC synapses (N = 12 neurons from 10 mice for 3 μM L-AP4, 8 neurons from 7 mice for 30 μM L-AP4 [data are from Fig.1A], and 5 neurons from 3 mice for 100 μM L-AP4. *P < 0.05 and **P < 0.01 by one-way ANOVA, [F(2,22)=7.30, P = 0.0037] with Bonferroni’s post-test. (B) The effect of LSP4-2022 on oEPSCs at thalamo-mPFC synapses. N = 6 neurons from 5 mice; *P < 0.05 by Wilcoxon matched-pairs test. (C) The effect of mGlu₈ agonist DCPG on oEPSCs. N = 5 neurons from 3 mice; P > 0.4 by Wilcoxon matched-pairs test. (D) The effect of mGlu₇ NAM ADX71743 with 30 μM L-AP4 on oEPSCs (N = 5 neurons from 3 mice) compared to the effect of 30 μM L-AP4 alone (same data as in Fig. 1A). P > 0.4 by Mann-Whitney test. (E and F) The effect of mGlu₄ PAM Lu AF21934, PHCCC or VU0418506 with 3 μM L-AP4 on oEPSCs (N = 6 neurons from 4 mice, 7 per 5, and 5 per 4, respectively) compared to the effect of 3 μM L-AP4 on oEPSCs (same data as in panel A). *P < 0.05 by one-way ANOVA [F(3,26) = 4.14, P = 0.0159] with Dunnett’s post-test. Calibration bars for oEPSC traces: 100pA/10ms, 100pA/10ms, 40pA/10ms (A); 100pA/10ms (B, C); 200pA/10ms (D); 100pA/10ms (E).

Fig. 3.. Effect of mGlu₂ NAM MRK-8-29 on L-AP4 induced inhibition of thalamo-mPFC synaptic transmission.

(A and B) The effects of MRK-8-29 on L-AP4 CRC in HEK cells expressing mGlu₄ homodimers (A) or mGlu_2/4 heterodimers (B) by GIRK assay. (C and D) The effects of MRK-8-29 on L-AP4 CRC in HEK cells expressing mGlu₄ homodimers (C) or mGlu_2/4 heterodimers (D) by CODA-RET assay. (E) The schematic diagrams illustrate the effect of MRK-8-29 on L-AP4 induced response at mGlu₄ homodimers (left) or mGlu_2/4 heterodimers (right). (F) The effect of 30 μM MRK-8-29 with L-AP4 on oEPSCs (N = 7 neurons from 5 mice) compared to the effect of 30 μM L-AP4 alone (same data as in Fig. 1A). ***P < 0.001 by Mann-Whitney test. Calibration bars for oEPSC traces: 100pA/10ms.

Fig. 4.. Effects of L-AP4 and MRK-8-29 on 5-HT₂ agonist DOI induced increase in sEPSCs in mPFC layer V pyramidal cells.

(A to C) Sample traces (top), cumulative probability plots of inter-event interval (IEI) of sEPSCs (middle) from three representative cells and summary plot of normalized group data (bottom) before and during application of 10μM DOI (A), before and during application of 10μM DOI in the presence of 3 μM L-AP4, and before and during application of 10μM DOI in the presence of 30 μM MRK-8-29 and 3 μM L-AP4. Calibration bars for sEPSC traces: 20pA/1s (A and B), and 30pA/1s (C). Normalized sEPSC frequency after application of 10μM DOI verse baseline under different conditions were compared using Wilcoxon matched-pairs tests: *P < 0.05, N = 7 neurons from 5 mice (A, bottom); P > 0.1, N = 5 per 4 (B, bottom); *P < 0.05, N = 6 per 4 (C, bottom). (D) Cumulative probability plots summarizing DOI-induced sEPSC frequency change in control, in the presence of L-AP4 and in the combination of L-AP4 and MRK-8-29. * P < 0.05 and ** P < 0.01 by one-way ANOVA [F(2,15)=7.113, P = 0.0067] with Bonferroni’s post-test.