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Differential activity of mGlu7 allosteric modulators provides evidence for mGlu7/8 heterodimers at hippocampal Schaffer collateral-CA1 synapses

  • Xin Lin
    Affiliations
    Department of Psychiatry, Vagelos College of Physicians and Surgeons, Columbia University, New York, New York, USA

    Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York, USA
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  • Nicole M. Fisher
    Affiliations
    Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA

    Warren Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, Tennessee, USA
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  • Shalini Dogra
    Affiliations
    Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA

    Warren Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, Tennessee, USA
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  • Rebecca K. Senter
    Affiliations
    Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA

    Warren Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, Tennessee, USA
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  • Carson W. Reed
    Affiliations
    Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA

    Warren Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, Tennessee, USA
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  • Jacob J. Kalbfleisch
    Affiliations
    Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA

    Warren Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, Tennessee, USA
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  • Craig W. Lindsley
    Affiliations
    Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA

    Warren Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, Tennessee, USA

    Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee, USA

    Department of Chemistry, Vanderbilt University, Nashville, Tennessee, USA
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  • Wesley B. Asher
    Affiliations
    Department of Psychiatry, Vagelos College of Physicians and Surgeons, Columbia University, New York, New York, USA

    Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York, USA
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  • Zixiu Xiang
    Affiliations
    Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA

    Warren Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, Tennessee, USA
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  • Colleen M. Niswender
    Correspondence
    For correspondence: Colleen M. Niswender; Jonathan A. Javitch
    Affiliations
    Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA

    Warren Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, Tennessee, USA

    Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee, USA

    Department of Chemistry, Vanderbilt University, Nashville, Tennessee, USA

    Vanderbilt Brain Institute, Vanderbilt University, Nashville, Tennessee, USA

    Vanderbilt Kennedy Center, Vanderbilt University Medical Center, Nashville, Tennessee, USA
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  • Jonathan A. Javitch
    Correspondence
    For correspondence: Colleen M. Niswender; Jonathan A. Javitch
    Affiliations
    Department of Psychiatry, Vagelos College of Physicians and Surgeons, Columbia University, New York, New York, USA

    Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York, USA

    Department of Molecular Pharmacology and Therapeutics, Vagelos College of Physicians and Surgeons, Columbia University, New York, New York, USA
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Open AccessPublished:September 02, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102458
      Glutamate acts at eight metabotropic glutamate (mGlu) receptor subtypes expressed in a partially overlapping fashion in distinct brain circuits. Recent evidence indicates that specific mGlu receptor protomers can heterodimerize and that these heterodimers can exhibit different pharmacology when compared to their homodimeric counterparts. Group III mGlu agonist-induced suppression of evoked excitatory potentials and induction of long-term potentiation at Schaffer collateral-CA1 (SC-CA1) synapses in the rodent hippocampus can be blocked by the selective mGlu7 negative allosteric modulator (NAM), ADX71743. Curiously, a different mGlu7 NAM, 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazonolo[4,5-c]pyridin-4(5H)-one, failed to block these responses in brain slices despite its robust activity at mGlu7 homodimers in vitro. We hypothesized that this might result from heterodimerization of mGlu7 with another mGlu receptor protomer and focused on mGlu8 as a candidate given the reported effects of mGlu8-targeted compounds in the hippocampus. Here, we used complemented donor acceptor-resonance energy transfer to study mGlu7/8 heterodimer activation in vitro and observed that ADX71743 blocked responses of both mGlu7/7 homodimers and mGlu7/8 heterodimers, whereas 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazonolo[4,5-c]pyridin-4(5H)-one only antagonized responses of mGlu7/7 homodimers. Taken together with our electrophysiology observations, these results suggest that a receptor with pharmacology consistent with an mGlu7/8 heterodimer modulates the activity of SC-CA1 synapses. Building on this hypothesis, we identified two additional structurally related mGlu7 NAMs that also differ in their activity at mGlu7/8 heterodimers, in a manner consistent with their ability to inhibit synaptic transmission and plasticity at SC-CA1. Thus, we propose that mGlu7/8 heterodimers are a key molecular target for modulating the activity of hippocampal SC-CA1 synapses.

      Keywords

      Abbreviations:

      ACSF (artificial cerebrospinal fluid), BRET (bioluminescence resonance energy transfer), CODA-RET (Complemented Donor Acceptor-Resonance Energy Transfer), fEPSPs (field excitatory postsynaptic potentials), GPCR (G protein-coupled receptor), HFS (high frequency stimulation), ITI (inter-train interval), LTP (long-term potentiation), mGlu (metabotropic glutamate receptor), mPFC (medial prefrontal cortex), mVenus (monomeric venus), NAM (negative allosteric modulator), NanoLuc (Nano luciferase), PAM (positive allosteric modulator), PBS (phosphate buffered saline), RLuc8 (Renilla luciferase 8), SC-CA1 (Schaffer Collateral-CA1), TBS (theta burst stimulation)
      Glutamate, the major excitatory neurotransmitter in the brain, acts at eight metabotropic glutamate (mGlu) receptors, all belonging to the G protein-coupled receptor (GPCR) family. These eight receptors are divided into three major groups based on sequence similarity, G protein coupling, and shared pharmacology (
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      Much of the work to date on mGlu receptor heterodimers has focused on mGlu2/4 heterodimers (
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      • Yin S.
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      • Zamorano R.
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      • et al.
      Selective actions of novel allosteric modulators reveal functional heteromers of metabotropic glutamate receptors in the CNS.
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      ). Since mGlu2 is co-expressed with mGlu4 at these synapses (
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      Metabotropic glutamate receptor-mediated presynaptic depression at corticostriatal synapses involves mGLuR2 or 3.
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      Group 2 metabotropic glutamate receptors induced long term depression in mouse striatal slices.
      ), we hypothesized that differential PAM activity at mGlu4/4 homodimers and mGlu2/4 heterodimers might underlie these electrophysiology results (
      • Yin S.
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      • Johnson K.A.
      • Zamorano R.
      • Jalan-Sakrikar N.
      • Gregory K.J.
      • et al.
      Selective actions of novel allosteric modulators reveal functional heteromers of metabotropic glutamate receptors in the CNS.
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      ). To test our hypothesis, we used complemented donor acceptor-resonance energy transfer (CODA-RET), an in vitro technique developed in our laboratory, to selectively measure signaling by defined heterodimers without contamination by homodimers expressed in the same cells (
      • Urizar E.
      • Yano H.
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      • Gales C.
      • Lambert N.
      • Javitch J.A.
      CODA-RET reveals functional selectivity as a result of GPCR heteromerization.
      ). CODA-RET revealed that mGlu4 PAMs were segregated into two categories: those that can enhance activity of the mGlu2/4 heterodimer, and those that cannot (
      • Niswender C.M.
      • Jones C.K.
      • Lin X.
      • Bubser M.
      • Thompson Gray A.
      • Blobaum A.L.
      • et al.
      Development and antiparkinsonian activity of VU0418506, a selective positive allosteric modulator of metabotropic glutamate receptor 4 homomers without activity at mGlu2/4 heteromers.
      ), which mirrored their ability to potentiate responses at corticostriatal synapses (
      • Yin S.
      • Noetzel M.J.
      • Johnson K.A.
      • Zamorano R.
      • Jalan-Sakrikar N.
      • Gregory K.J.
      • et al.
      Selective actions of novel allosteric modulators reveal functional heteromers of metabotropic glutamate receptors in the CNS.
      ). Furthermore, by pairing electrophysiological recordings at various cortical inputs with additional CODA-RET studies, we were able to establish a critical role for mGlu2/4 heterodimers at projections from the thalamus to the medial prefrontal cortex (mPFC), but not at hippocampal–mPFC or amygdala–mPFC synapses (
      • Xiang Z.
      • Lv X.
      • Lin X.
      • O'Brien D.E.
      • Altman M.K.
      • Lindsley C.W.
      • et al.
      Input-specific regulation of glutamatergic synaptic transmission in the medial prefrontal cortex by mGlu2/mGlu4 receptor heterodimers.
      ), suggesting differences in expression of various homodimer and heterodimer pairs that could eventually be exploited therapeutically.
      In addition to mGlu2 and mGlu4, Doumazane et al. (
      • Doumazane E.
      • Scholler P.
      • Zwier J.M.
      • Trinquet E.
      • Rondard P.
      • Pin J.P.
      A new approach to analyze cell surface protein complexes reveals specific heterodimeric metabotropic glutamate receptors.
      ) demonstrated that all group II and group III mGlu receptors can heterodimerize in vitro. The group III mGlu receptors primarily act as presynaptic autoreceptors and heteroreceptors (
      • Niswender C.M.
      • Conn P.J.
      Metabotropic glutamate receptors: physiology, pharmacology, and disease.
      ); among this subgroup, mGlu7 and mGlu8 are co-expressed in many of the same brain regions, including the hippocampus (
      • Corti C.
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      • Brabet I.
      • Corsi M.
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      Cloning and characterization of alternative mRNA forms for the rat metabotropic glutamate receptors mGluR7 and mGluR8.
      ). Using group III mGlu-specific agonists such as L-AP4, pharmacological profiles consistent with a role for both mGlu7 and mGlu8 at Schaffer collateral-CA1 (SC-CA1) synapses have been identified (
      • Ayala J.E.
      • Niswender C.M.
      • Luo Q.
      • Banko J.L.
      • Conn P.J.
      Group III mGluR regulation of synaptic transmission at the SC-CA1 synapse is developmentally regulated.
      ,
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      • Engers D.W.
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      • et al.
      Activation of metabotropic glutamate receptor 7 is required for induction of long-term potentiation at SC-CA1 synapses in the hippocampus.
      ,
      • Baskys A.
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      Agonists at metabotropic glutamate receptors presynaptically inhibit EPSCs in neonatal rat hippocampus.
      ). In these studies, after L-AP4 application, field excitatory postsynaptic potentials (fEPSPs) were reduced, while paired pulse ratios increased, suggesting a presynaptic mechanism (
      • Ayala J.E.
      • Niswender C.M.
      • Luo Q.
      • Banko J.L.
      • Conn P.J.
      Group III mGluR regulation of synaptic transmission at the SC-CA1 synapse is developmentally regulated.
      ,
      • Klar R.
      • Walker A.G.
      • Ghose D.
      • Grueter B.A.
      • Engers D.W.
      • Hopkins C.R.
      • et al.
      Activation of metabotropic glutamate receptor 7 is required for induction of long-term potentiation at SC-CA1 synapses in the hippocampus.
      ,
      • Somogyi P.
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      • Lujan R.
      • Roberts J.D.
      • Watanabe M.
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      High level of mGluR7 in the presynaptic active zones of select populations of GABAergic terminals innervating interneurons in the rat hippocampus.
      ). Notably, of the other group III receptors, mGlu6 expression is restricted to the retina (
      • Nakajima Y.
      • Iwakabe H.
      • Akazawa C.
      • Nawa H.
      • Shigemoto R.
      • Mizuno N.
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      Molecular characterization of a novel retinal metabotropic glutamate receptor mGluR6 with a high agonist selectivity for L-2-amino-4-phosphonobutyrate.
      ), and although L-AP4 can activate mGlu4, an mGlu4-selective PAM and an mGlu4-preferring agonist did not affect fEPSPs measured at SC-CA1 synapses (
      • Ayala J.E.
      • Niswender C.M.
      • Luo Q.
      • Banko J.L.
      • Conn P.J.
      Group III mGluR regulation of synaptic transmission at the SC-CA1 synapse is developmentally regulated.
      ). Additionally, use of a different and recently described group III agonist, LSP4-2022 (
      • Goudet C.
      • Vilar B.
      • Courtiol T.
      • Deltheil T.
      • Bessiron T.
      • Brabet I.
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      A novel selective metabotropic glutamate receptor 4 agonist reveals new possibilities for developing subtype selective ligands with therapeutic potential.
      ), in a concentration range that is selective for mGlu4 over mGlu7 and mGlu8, did not affect fEPSPs (
      • Klar R.
      • Walker A.G.
      • Ghose D.
      • Grueter B.A.
      • Engers D.W.
      • Hopkins C.R.
      • et al.
      Activation of metabotropic glutamate receptor 7 is required for induction of long-term potentiation at SC-CA1 synapses in the hippocampus.
      ). While a group II mGlu receptor agonist can induce long-term potentiation (LTP) at SC-CA1 synapses, this effect has recently been shown to be mediated by a novel postsynaptic mechanism (
      • Rosenberg N.
      • Gerber U.
      • Ster J.
      Activation of group II metabotropic glutamate receptors promotes LTP induction at Schaffer collateral-CA1 pyramidal cell synapses by priming NMDA receptors.
      ). Collectively, these results suggest that presynaptic responses at SC-CA1 synapses are most likely mediated by mGlu7 and/or mGlu8.
      To explore this question further, we focused on an evaluation of multiple mGlu7-selective negative allosteric modulators (NAMs) at SC-CA1 synapses (
      • Suzuki G.
      • Tsukamoto N.
      • Fushiki H.
      • Kawagishi A.
      • Nakamura M.
      • Kurihara H.
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      In vitro pharmacological characterization of novel isoxazolopyridone derivatives as allosteric metabotropic glutamate receptor 7 antagonists.
      ,
      • Kalinichev M.
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      • Royer-Urios I.
      • Bournique B.
      • Finn T.
      • et al.
      ADX71743, a potent and selective negative allosteric modulator of metabotropic glutamate receptor 7: in vitro and in vivo characterization.
      ,
      • Reed C.W.
      • McGowan K.M.
      • Spearing P.K.
      • Stansley B.J.
      • Roenfanz H.F.
      • Engers D.W.
      • et al.
      VU6010608, a novel mGlu7 NAM from a series of N-(2-(1H-1,2,4-Triazol-1-yl)-5-(trifluoromethoxy)phenyl)benzamides.
      ). Two of these NAMs, ADX71743 and 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazonolo[4,5-c]pyridin-4(5H)-one (MMPIP), block responses in heterologous cells that express only mGlu7 homodimers (
      • Suzuki G.
      • Tsukamoto N.
      • Fushiki H.
      • Kawagishi A.
      • Nakamura M.
      • Kurihara H.
      • et al.
      In vitro pharmacological characterization of novel isoxazolopyridone derivatives as allosteric metabotropic glutamate receptor 7 antagonists.
      ,
      • Kalinichev M.
      • Rouillier M.
      • Girard F.
      • Royer-Urios I.
      • Bournique B.
      • Finn T.
      • et al.
      ADX71743, a potent and selective negative allosteric modulator of metabotropic glutamate receptor 7: in vitro and in vivo characterization.
      ), and ADX71743 has been reported to block agonist-mediated inhibition of fEPSPs at SC-CA1 synapses (
      • Kalinichev M.
      • Rouillier M.
      • Girard F.
      • Royer-Urios I.
      • Bournique B.
      • Finn T.
      • et al.
      ADX71743, a potent and selective negative allosteric modulator of metabotropic glutamate receptor 7: in vitro and in vivo characterization.
      ), an effect we replicated in the study by Klar et al. (
      • Klar R.
      • Walker A.G.
      • Ghose D.
      • Grueter B.A.
      • Engers D.W.
      • Hopkins C.R.
      • et al.
      Activation of metabotropic glutamate receptor 7 is required for induction of long-term potentiation at SC-CA1 synapses in the hippocampus.
      ). In contrast, we showed that MMPIP did not block L-AP4-induced effect on fEPSPs at this same synapse (
      • Niswender C.M.
      • Johnson K.A.
      • Miller N.R.
      • Ayala J.E.
      • Luo Q.
      • Williams R.
      • et al.
      Context-dependent pharmacology exhibited by negative allosteric modulators of metabotropic glutamate receptor 7.
      ). Here, we show that this divergence in activity between ADX71743 and MMPIP also extends to LTP. Using CODA-RET, we now show that ADX71743 blocks agonist-mediated responses at mGlu7/8 heterodimers, whereas MMPIP is without effect. We extend these findings to two highly structurally related mGlu7 NAMs, VU6010608 and VU6010953 (
      • Reed C.W.
      • McGowan K.M.
      • Spearing P.K.
      • Stansley B.J.
      • Roenfanz H.F.
      • Engers D.W.
      • et al.
      VU6010608, a novel mGlu7 NAM from a series of N-(2-(1H-1,2,4-Triazol-1-yl)-5-(trifluoromethoxy)phenyl)benzamides.
      ,
      • Reed C.W.
      • Rodriguez A.L.
      • Kalbfleisch J.J.
      • Seto M.
      • Jenkins M.T.
      • Blobaum A.L.
      • et al.
      Development and profiling of mGlu7 NAMs with a range of saturable inhibition of agonist responses in vitro.
      ), which are structurally identical except for different alkoxy substitutions; these two compounds also show differential activity at mGlu7 homodimers and mGlu7/8 heterodimers that match their profiles in blocking LTP at SC-CA1 synapses. These studies suggest that the complexity of mGlu receptor assembly has widespread implications for receptor pharmacology and, by extension, therapeutic targeting.

      Results

      The mGlu7 NAM MMPIP does not block LTP at SC-CA1

      We have previously identified a divergence in pharmacology of two mGlu7 NAMs, ADX71743 and MMPIP (Fig. 1A), in blocking L-AP4’s effect on fEPSPs at SC-CA1 (
      • Klar R.
      • Walker A.G.
      • Ghose D.
      • Grueter B.A.
      • Engers D.W.
      • Hopkins C.R.
      • et al.
      Activation of metabotropic glutamate receptor 7 is required for induction of long-term potentiation at SC-CA1 synapses in the hippocampus.
      ,
      • Kalinichev M.
      • Rouillier M.
      • Girard F.
      • Royer-Urios I.
      • Bournique B.
      • Finn T.
      • et al.
      ADX71743, a potent and selective negative allosteric modulator of metabotropic glutamate receptor 7: in vitro and in vivo characterization.
      ,
      • Niswender C.M.
      • Johnson K.A.
      • Miller N.R.
      • Ayala J.E.
      • Luo Q.
      • Williams R.
      • et al.
      Context-dependent pharmacology exhibited by negative allosteric modulators of metabotropic glutamate receptor 7.
      ), despite both compounds blocking mGlu7 homodimer-mediated responses in vitro (Fig. 1B). We have also previously shown that ADX71743 blocks the induction of LTP at SC-CA1 synapses (
      • Klar R.
      • Walker A.G.
      • Ghose D.
      • Grueter B.A.
      • Engers D.W.
      • Hopkins C.R.
      • et al.
      Activation of metabotropic glutamate receptor 7 is required for induction of long-term potentiation at SC-CA1 synapses in the hippocampus.
      ). In contrast, but consistent with its lack of effect on L-AP4-mediated inhibition of fEPSPs (
      • Niswender C.M.
      • Johnson K.A.
      • Miller N.R.
      • Ayala J.E.
      • Luo Q.
      • Williams R.
      • et al.
      Context-dependent pharmacology exhibited by negative allosteric modulators of metabotropic glutamate receptor 7.
      ), MMPIP does not block LTP at SC-CA1 (Fig. 1, C and D).
      Figure thumbnail gr1
      Figure 1Despite robust blockade of mGlu7/7 homodimer activity in vitro, MMPIP does not block LTP at SC-CA1 synapses. A, the structures of ADX71743 and MMPIP are shown. B, increasing concentrations of the mGlu7 NAMs ADX71743 (orange) and MMPIP (dark red) were applied to HEK293 cells expressing rat mGlu7 and the promiscuous G protein, Gα15. Both compounds were able to inhibit L-AP4-induced calcium responses with mean IC50s of 460 and 72 nM, respectively (mean ± SEM, n = 5 or 3 independent determinations in triplicate, respectively). C, electrophysiology experiments showing the effect of vehicle (black), 3 μM ADX71743 (orange), or 10 μM MMPIP (dark red) applied to brain slices 20 min prior (black bar) to the induction of LTP at SC-CA1 synapses using an HFS protocol. Data represent the mean ± SEM of 3 to 5 slices per condition. Data for ADX71743 originally appeared in the study by Klar et al. (
      • Klar R.
      • Walker A.G.
      • Ghose D.
      • Grueter B.A.
      • Engers D.W.
      • Hopkins C.R.
      • et al.
      Activation of metabotropic glutamate receptor 7 is required for induction of long-term potentiation at SC-CA1 synapses in the hippocampus.
      ). D, the average of the last 5 min of recording in C (gray box) is plotted for vehicle (black), ADX71743 (orange), and MMPIP (dark red). One-way ANOVA with Tukey’s post hoc test, ∗∗p < 0.01, ∗∗∗p < 0.001. HFS, high-frequency stimulation; LTP, long-term potentiation; NAM, negative allosteric modulator; SC-CA1, Schaffer collateral-CA1.

      ADX71743 and MMPIP differentially inhibit mGlu7/8 heterodimers

      Given that ADX71743 and MMPIP share the ability to block the activity of mGlu7 homodimers in vitro, it was unexpected to observe such distinct effects on electrophysiological measures at SC-CA1. To explore the potential for differential activity of the two NAMs at mGlu7/7 homodimers and mGlu7/8 heterodimers, we used CODA-RET (Fig. 2, AC) (
      • Urizar E.
      • Yano H.
      • Kolster R.
      • Gales C.
      • Lambert N.
      • Javitch J.A.
      CODA-RET reveals functional selectivity as a result of GPCR heteromerization.
      ). To carry out this assay, mGlu7 and mGlu8 were fused at their C-termini with split luciferase fragments. These two fragments (L1 and L2) are incapable of producing bioluminescence when expressed alone, but when brought into proximity, they complement to form a functional enzyme capable of generating bioluminescence (
      • Paulmurugan R.
      • Umezawa Y.
      • Gambhir S.S.
      Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies.
      ,
      • Lund C.H.
      • Bromley J.R.
      • Stenbaek A.
      • Rasmussen R.E.
      • Scheller H.V.
      • Sakuragi Y.
      A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus.
      ). By monitoring bioluminescence resonance energy transfer (BRET) between the complemented luciferase (donor), which identifies the pair of mGlu protomers, and monomeric Venus (mVenus) (acceptor)-labeled Gαi subunits, we can selectively measure signaling by defined heterodimers (Fig. 2B) or homodimers (Fig. 2, A and C). Using CODA-RET, we found that in cells expressing mGlu7/7 homodimers, the potency of DL-AP4 was 100-fold lower than that observed for mGlu7/8 heterodimers (Fig. 2, D and E). Furthermore, in cells expressing mGlu7/7 homodimers, both ADX71743 and MMPIP antagonized agonist-induced responses (Fig. 2D). Conversely, in cells expressing mGlu7/8 heterodimers, only ADX71743 blocked the CODA-RET signal (Fig. 2E). As expected, ADX71743 was inactive at mGlu8/8 homodimers (Fig. 2F), consistent with its reported specificity for mGlu7 (
      • Kalinichev M.
      • Rouillier M.
      • Girard F.
      • Royer-Urios I.
      • Bournique B.
      • Finn T.
      • et al.
      ADX71743, a potent and selective negative allosteric modulator of metabotropic glutamate receptor 7: in vitro and in vivo characterization.
      ).
      Figure thumbnail gr2
      Figure 2ADX71743 and MMPIP differentially inhibit mGlu7/8 heterodimers. AC, schematics of the CODA-RET approach. The defined (B) mGlu7/8 heterodimer and (A) mGlu7/7 and (C) mGlu8/8 homodimers are shown with complemented split fragments (L1 and L2) of the luciferase donor (L1:L2), leading to luminescence and BRET-based CODA-RET via the Gαi-fused mVenus upon receptor activation. As shown, homodimers that are formed by protomers fused to noncomplementing luciferase fragments will not luminesce and, therefore, do not contribute to the BRET signal. Note that while the split fragments are all conceptually denoted here as L1 or L2, we used split RLuc8 for (A) and (B) and split Nanoluc for (C), as described in . DF, CODA-RET results showing DL-AP4-concentration response curves in the presence of 50 μM of the indicated mGlu7 NAMs for (D) mGlu7/7 homodimers, (E) mGlu7/8 heterodimers, and (F) mGlu8/8 homodimers, respectively. Note that the mGlu7 preferring NAM ADX71743 is active at mGlu7/7 and mGlu7/8 but inactive at mGlu8/8, as expected, whereas MMPIP is only active at mGlu7/7. Error bars represent the mean ± SEM for at least three independent experiments performed in triplicate. BRET, bioluminescence resonance energy transfer; CODA-RET, complemented donor acceptor-resonance energy transfer; NAM, negative allosteric modulator.

      Two highly similar NAMs, VU6010608 and VU6010953, show differential blockade of LTP at SC-CA1 synapses that is consistent with activity at mGlu7/8 heterodimers

      During our medicinal chemistry campaign to optimize allosteric modulators of mGlu7, we recently identified VU6010608 and VU6010953, compounds that differ structurally by a single alkoxy substitution and possess highly similar in vitro profiles in cells expressing mGlu7 homodimers (
      • Reed C.W.
      • McGowan K.M.
      • Spearing P.K.
      • Stansley B.J.
      • Roenfanz H.F.
      • Engers D.W.
      • et al.
      VU6010608, a novel mGlu7 NAM from a series of N-(2-(1H-1,2,4-Triazol-1-yl)-5-(trifluoromethoxy)phenyl)benzamides.
      ,
      • Reed C.W.
      • Rodriguez A.L.
      • Kalbfleisch J.J.
      • Seto M.
      • Jenkins M.T.
      • Blobaum A.L.
      • et al.
      Development and profiling of mGlu7 NAMs with a range of saturable inhibition of agonist responses in vitro.
      ) (Fig. 3A). We have previously shown that VU6010608 blocks high-frequency stimulation (HFS)–induced LTP at SC-CA1 (
      • Reed C.W.
      • McGowan K.M.
      • Spearing P.K.
      • Stansley B.J.
      • Roenfanz H.F.
      • Engers D.W.
      • et al.
      VU6010608, a novel mGlu7 NAM from a series of N-(2-(1H-1,2,4-Triazol-1-yl)-5-(trifluoromethoxy)phenyl)benzamides.
      ), and we show here that it was also effective in blocking LTP induced using an alternate stimulation protocol, theta burst stimulation (TBS) (Fig. 3, B and C). To our surprise, the highly related VU6010953 compound failed to block TBS-induced LTP at the same synapses (Fig. 3, B and C). An examination of the profile of these two compounds using CODA-RET showed that both completely blocked responses to mGlu7/7 homodimers (Fig. 3D), but, like ADX71743 and MMPIP, they diverged in their activity at mGlu7/8 heterodimers (Fig. 3E), with VU6010953 unable to inhibit activity of the heterodimer, consistent with its lack of effect on LTP. VU6010608 was inactive at mGlu8/8 in CODA-RET (Fig. 3F), confirming its reported preference for mGlu7 (
      • Reed C.W.
      • McGowan K.M.
      • Spearing P.K.
      • Stansley B.J.
      • Roenfanz H.F.
      • Engers D.W.
      • et al.
      VU6010608, a novel mGlu7 NAM from a series of N-(2-(1H-1,2,4-Triazol-1-yl)-5-(trifluoromethoxy)phenyl)benzamides.
      ).
      Figure thumbnail gr3
      Figure 3The NAMs VU6010608 and VU6010953 diverge in blocking responses at SC-CA1 synapses and at mGlu7/8 heterodimers as assessed by CODA-RET. A, the structures of VU6010608 and VU6010953 are shown, with the different alkoxy substitutions shown in blue (VU6010608: methoxy; VU6010953: propoxy), along with their activities (IC50 values from references (
      • Reed C.W.
      • McGowan K.M.
      • Spearing P.K.
      • Stansley B.J.
      • Roenfanz H.F.
      • Engers D.W.
      • et al.
      VU6010608, a novel mGlu7 NAM from a series of N-(2-(1H-1,2,4-Triazol-1-yl)-5-(trifluoromethoxy)phenyl)benzamides.
      ,
      • Reed C.W.
      • Rodriguez A.L.
      • Kalbfleisch J.J.
      • Seto M.
      • Jenkins M.T.
      • Blobaum A.L.
      • et al.
      Development and profiling of mGlu7 NAMs with a range of saturable inhibition of agonist responses in vitro.
      )) in blocking rat mGlu7-Gα15-mediated calcium responses. B, electrophysiology experiments showing that bath application of 10 μM VU6010608 (green diamonds) to brain slices for 10 min prior to TBS (black bar) blocked LTP at SC-CA1 synapses compared to vehicle (black circles). In contrast, application of 30 μM VU6010953 (cyan hexagons) did not block TBS-induced LTP at the same synapses. C, the average of the last 5 min of recording in B (gray box) is plotted for vehicle (black), VU6010608 (VU608, green), and VU6010953 (VU953, cyan). Data represent the mean ± SEM of 4 to 15 slices per condition analyzed using one way ANOVA with a Tukey’s post hoc test; ∗p < 0.05. DF, CODA-RET results showing DL-AP4-concentration response curves in the presence of 50 μM of the indicated mGlu7 NAMs for (D) mGlu7/7 homodimers, (E) mGlu7/8 heterodimers, and (F) mGlu8/8 homodimers. Note that the NAM VU6010608 is active at mGlu7/7 and mGlu7/8 but inactive at mGlu8/8, as expected, whereas VU6010953 is only active at mGlu7/7. Error bars represent the mean ± SEM for at least three independent experiments performed in triplicate. CODA-RET, complemented donor acceptor-resonance energy transfer; LTP, long-term potentiation; NAM, negative allosteric modulator; SC-CA1, Schaffer collateral-CA1; TBS, theta burst stimulation.

      Discussion

      Glutamate exerts critical actions at a variety of mGlu receptors that are differentially expressed in various circuits throughout the brain, making them highly attractive targets for novel therapeutics. However, such efforts can be complicated by the expression of the same receptor in multiple brain regions, making it challenging to avoid off-target effects. GPCR heterodimers have long been touted as potential targets to enhance the specificity of drug action, but there has been relatively little evidence for their expression in vivo. Emerging evidence for the expression and activity of mGlu2/4 heterodimers at certain synapses, but not others (
      • Yin S.
      • Noetzel M.J.
      • Johnson K.A.
      • Zamorano R.
      • Jalan-Sakrikar N.
      • Gregory K.J.
      • et al.
      Selective actions of novel allosteric modulators reveal functional heteromers of metabotropic glutamate receptors in the CNS.
      ,
      • Xiang Z.
      • Lv X.
      • Lin X.
      • O'Brien D.E.
      • Altman M.K.
      • Lindsley C.W.
      • et al.
      Input-specific regulation of glutamatergic synaptic transmission in the medial prefrontal cortex by mGlu2/mGlu4 receptor heterodimers.
      ,
      • Moreno Delgado D.
      • Moller T.C.
      • Ster J.
      • Giraldo J.
      • Maurel D.
      • Rovira X.
      • et al.
      Pharmacological evidence for a metabotropic glutamate receptor heterodimer in neuronal cells.
      ,
      • Liu J.
      • Zhang Z.
      • Moreno-Delgado D.
      • Dalton J.A.
      • Rovira X.
      • Trapero A.
      • et al.
      Allosteric control of an asymmetric transduction in a G protein-coupled receptor heterodimer.
      ,
      • Meng J.
      • Xu C.
      • Lafon P.A.
      • Roux S.
      • Mathieu M.
      • Zhou R.
      • et al.
      Nanobody-based sensors reveal a high proportion of mGlu heterodimers in the brain.
      ), has created an opportunity to differentiate homodimers and heterodimers pharmacologically, providing exciting precedent for this approach.
      We show here that the pharmacology of select mGlu7 receptor ligands at the SC-CA1 synapse is not consistent with that of an mGlu7 homodimer, as ADX71743 inhibits both group III agonist-induced effects on fEPSPs and LTP, whereas MMPIP, which is a fully efficacious NAM at mGlu7 homodimers in vitro, is completely without activity at SC-CA1 in brain slices. We hypothesized that this might result from heterodimerization of mGlu7 with another presynaptic partner. mGlu6 is restricted in expression to the retina (
      • Nakajima Y.
      • Iwakabe H.
      • Akazawa C.
      • Nawa H.
      • Shigemoto R.
      • Mizuno N.
      • et al.
      Molecular characterization of a novel retinal metabotropic glutamate receptor mGluR6 with a high agonist selectivity for L-2-amino-4-phosphonobutyrate.
      ), and, because the observed pharmacology in previous electrophysiology experiments argues against the involvement of mGlu4 (
      • Ayala J.E.
      • Niswender C.M.
      • Luo Q.
      • Banko J.L.
      • Conn P.J.
      Group III mGluR regulation of synaptic transmission at the SC-CA1 synapse is developmentally regulated.
      ,
      • Klar R.
      • Walker A.G.
      • Ghose D.
      • Grueter B.A.
      • Engers D.W.
      • Hopkins C.R.
      • et al.
      Activation of metabotropic glutamate receptor 7 is required for induction of long-term potentiation at SC-CA1 synapses in the hippocampus.
      ), we turned to the other widely expressed presynaptic group III mGlu receptor, mGlu8, as a potential mGlu7 partner in the SC-CA1 area of the hippocampus. Given that the mGlu8 receptor is expressed in the hippocampus and that the mGlu8 agonist DCPG has been shown to act in this region (
      • Ayala J.E.
      • Niswender C.M.
      • Luo Q.
      • Banko J.L.
      • Conn P.J.
      Group III mGluR regulation of synaptic transmission at the SC-CA1 synapse is developmentally regulated.
      ,
      • Baskys A.
      • Malenka R.C.
      Agonists at metabotropic glutamate receptors presynaptically inhibit EPSCs in neonatal rat hippocampus.
      ), we hypothesized that mGlu7/8 heterodimerization might explain these native tissue findings. Our CODA-RET results were completely consistent with this hypothesis, as ADX71743 acted as a NAM at mGlu7/7 and mGlu7/8 heterodimers, whereas MMPIP was active at mGlu7/7 but without effect at mGlu7/8 receptors. Remarkably, we also found that members of a distinct structural scaffold could also differentiate mGlu7/7 homodimers and mGlu7/8 heterodimers, with VU6010953 inactive at mGlu7/8 heterodimers as assessed by CODA-RET and ineffective at blocking LTP at SC-CA1 synapses. In contrast, VU6010608, which differs from VU6010953 only by a single alkoxy moiety, was active both in vitro at mGlu7/8 heterodimers and in brain slices. That such a small difference in the structure of these NAMs produced such a profound change in their activity is quite extraordinary. The impact on mGlu receptor pharmacology controlled by a single alkoxy moiety suggests an enormous potential richness in the pharmacology of these targets, which must be explored by new, more focused structure–activity relationship studies as well as structural experiments comparing homodimeric and heterodimeric combinations to begin to understand how allosteric propagation of receptor activity can differ so profoundly between various receptor combinations. mGlu receptor compounds characterized to date have been identified by their activity at mGlu receptor homodimers. Thus, while compounds can be identified serendipitously as also active at mGlu receptor heterodimers as we have done here, by design, ligands will also be active at the receptor homodimer combination used for their original identification. Future efforts to identify heterodimer-selective compounds will require rescreening of existing libraries of compounds using, for example, a CODA-RET heterodimer configuration, and then counter-screening against homodimers to remove compounds that act at both.
      Our findings strongly support the presence of mGlu7/8 heterodimers in modulating activity at hippocampal SC-CA1 synapses. Our previous finding that mGlu7 is required for the induction of LTP at these synapses (
      • Klar R.
      • Walker A.G.
      • Ghose D.
      • Grueter B.A.
      • Engers D.W.
      • Hopkins C.R.
      • et al.
      Activation of metabotropic glutamate receptor 7 is required for induction of long-term potentiation at SC-CA1 synapses in the hippocampus.
      ) suggests the potential for an mGlu7/8 heterodimer to contribute to hippocampal synaptic plasticity, learning, and memory. Historically, mGlu7 has been proposed to act as an “emergency brake” due to its low affinity for glutamate (
      • Niswender C.M.
      • Conn P.J.
      Metabotropic glutamate receptors: physiology, pharmacology, and disease.
      ); the confirmation that mGlu7-containing heterodimers exhibit dramatic left-shifts in agonist potency (
      • Habrian C.H.
      • Levitz J.
      • Vyklicky V.
      • Fu Z.
      • Hoagland A.
      • McCort-Tranchepain I.
      • et al.
      Conformational pathway provides unique sensitivity to a synaptic mGluR.
      ), however, suggests that this property may be specific to mGlu7/7 homodimers. We and others have shown that mGlu7 knockout animals, as well as animals modeling a loss-of-function mutation in mGlu7 found in patients with neurodevelopmental disorders (
      • Fisher N.M.
      • AlHashim A.
      • Buch A.B.
      • Badivuku H.
      • Samman M.M.
      • Weiss K.M.
      • et al.
      A GRM7 mutation associated with developmental delay reduces mGlu7 expression and produces neurological phenotypes.
      ), exhibit seizures that involve the hippocampus (
      • Fisher N.M.
      • Gould R.W.
      • Gogliotti R.G.
      • McDonald A.J.
      • Badivuku H.
      • Chennareddy S.
      • et al.
      Phenotypic profiling of mGlu7 knockout mice reveals new implications for neurodevelopmental disorders.
      ,
      • Fisher N.M.
      • Seto M.
      • Lindsley C.W.
      • Niswender C.M.
      Metabotropic glutamate receptor 7: a new therapeutic target in neurodevelopmental disorders.
      ,
      • Gogliotti R.G.
      • Senter R.K.
      • Fisher N.M.
      • Adams J.
      • Zamorano R.
      • Walker A.G.
      • et al.
      mGlu7 potentiation rescues cognitive, social, and respiratory phenotypes in a mouse model of Rett syndrome.
      ,
      • Sansig G.
      • Bushell T.J.
      • Clarke V.R.
      • Rozov A.
      • Burnashev N.
      • Portet C.
      • et al.
      Increased seizure susceptibility in mice lacking metabotropic glutamate receptor 7.
      ). Moreover, an agonist with mGlu7 activity has been shown to protect mice from the development and manifestation of seizures (
      • Girard B.
      • Tuduri P.
      • Moreno M.P.
      • Sakkaki S.
      • Barboux C.
      • Bouschet T.
      • et al.
      The mGlu7 receptor provides protective effects against epileptogenesis and epileptic seizures.
      ), and mGlu7 activation or potentiation has been considered as a novel strategy for the treatment of intellectual disability and epilepsy (reviewed in the study by Fisher et al. (
      • Fisher N.M.
      • Seto M.
      • Lindsley C.W.
      • Niswender C.M.
      Metabotropic glutamate receptor 7: a new therapeutic target in neurodevelopmental disorders.
      )). It is also noteworthy that the NAM ADX71743 has been shown to elicit seizures in animals (
      • Tassin V.
      • Girard B.
      • Chotte A.
      • Fontanaud P.
      • Rigault D.
      • Kalinichev M.
      • et al.
      Phasic and tonic mGlu7 receptor activity modulates the thalamocortical network.
      ); in contrast, MMPIP does not exacerbate seizures induced by electrical shock or potentiate pentylenetetrazole-induced seizures (
      • Hikichi H.
      • Murai T.
      • Okuda S.
      • Maehara S.
      • Satow A.
      • Ise S.
      • et al.
      Effects of a novel metabotropic glutamate receptor 7 negative allosteric modulator, 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazonolo[4,5-c]pyridin-4(5H)-one (MMPIP), on the central nervous system in rodents.
      ). The intriguing observation that these two NAMs differ at the level of the mGlu7/8 heterodimer suggests that future studies could explore the possibility that mGlu7/8 heterodimers mediate seizure activity, requiring further evaluation of how reductions or loss of mGlu7 in mice and humans causes seizure activity. Additionally, the finding that all of the group II and group III mGlu receptors can heterodimerize (
      • Doumazane E.
      • Scholler P.
      • Zwier J.M.
      • Trinquet E.
      • Rondard P.
      • Pin J.P.
      A new approach to analyze cell surface protein complexes reveals specific heterodimeric metabotropic glutamate receptors.
      ) suggests that it will now be essential to evaluate the profile of these two compounds, as well as other mGlu7 PAMs and NAMs, at various heterodimeric combinations using CODA-RET to provide additional context to pharmacological profiles observed at native tissue locations in which mGlu7 is co-expressed with other mGlu receptors. Based on our findings presented here, we anticipate that an evaluation of existing mGlu7 and mGlu8 orthosteric and allosteric ligands for activity at mGlu7/8 heterodimers will shed new light on the ideal pharmacological profile of therapeutic candidates.

      Experimental procedures

      Compounds

      L-AP4, DL-AP4, MMPIP, and glutamate were purchased from Tocris. LSP4-2022 and ADX71743 were synthesized in house using methods reported in the study by Klar et al. (
      • Klar R.
      • Walker A.G.
      • Ghose D.
      • Grueter B.A.
      • Engers D.W.
      • Hopkins C.R.
      • et al.
      Activation of metabotropic glutamate receptor 7 is required for induction of long-term potentiation at SC-CA1 synapses in the hippocampus.
      ). VU06010608 and VU6010953 were synthesized in house using methods reported in the study by Reed et al. (
      • Reed C.W.
      • McGowan K.M.
      • Spearing P.K.
      • Stansley B.J.
      • Roenfanz H.F.
      • Engers D.W.
      • et al.
      VU6010608, a novel mGlu7 NAM from a series of N-(2-(1H-1,2,4-Triazol-1-yl)-5-(trifluoromethoxy)phenyl)benzamides.
      ,
      • Reed C.W.
      • Rodriguez A.L.
      • Kalbfleisch J.J.
      • Seto M.
      • Jenkins M.T.
      • Blobaum A.L.
      • et al.
      Development and profiling of mGlu7 NAMs with a range of saturable inhibition of agonist responses in vitro.
      ).

      Calcium assays

      Calcium assays in which rat mGlu7 was coupled to calcium mobilization via the promiscuous G protein Gα15 were used to determine in vitro potency and efficacy and were conducted as described in (
      • Klar R.
      • Walker A.G.
      • Ghose D.
      • Grueter B.A.
      • Engers D.W.
      • Hopkins C.R.
      • et al.
      Activation of metabotropic glutamate receptor 7 is required for induction of long-term potentiation at SC-CA1 synapses in the hippocampus.
      ,
      • Reed C.W.
      • McGowan K.M.
      • Spearing P.K.
      • Stansley B.J.
      • Roenfanz H.F.
      • Engers D.W.
      • et al.
      VU6010608, a novel mGlu7 NAM from a series of N-(2-(1H-1,2,4-Triazol-1-yl)-5-(trifluoromethoxy)phenyl)benzamides.
      ,
      • Reed C.W.
      • Kalbfleisch J.J.
      • Wong M.J.
      • Washecheck J.P.
      • Hunter A.
      • Rodriguez A.L.
      • et al.
      Discovery of VU6027459: a first-in-class selective and CNS penetrant mGlu7 positive allosteric modulator tool compound.
      ,
      • Reed C.W.
      • Washecheck J.P.
      • Quitlag M.C.
      • Jenkins M.T.
      • Rodriguez A.L.
      • Engers D.W.
      • et al.
      Surveying heterocycles as amide bioisosteres within a series of mGlu7 NAMs: discovery of VU6019278.
      ,
      • Reed C.W.
      • Yohn S.E.
      • Washecheck J.P.
      • Roenfanz H.F.
      • Quitalig M.C.
      • Luscombe V.B.
      • et al.
      Discovery of an orally bioavailable and central nervous system (CNS) penetrant mGlu7 negative allosteric modulator (NAM) in vivo tool compound: N-(2-(1 H-1,2,4-triazol-1-yl)-5-(trifluoromethoxy)phenyl)-4-(cyclopropylmethoxy)-3-methox ybenzamide (VU6012962).
      ).

      Construction and transfection of expression vectors for CODA-RET assays

      cDNAs for rat mGlu7 and mGlu8 were N-terminally tagged with a hemagglutinin epitope tag using standard molecular biology procedures. cDNAs encoding the split fragments of Renilla Luciferase 8, L1 (residues 1–229), or L2 (residues 230–311), were fused in frame to the C-terminus of mGlu7 and mGlu8 following the linker “GSPPARAT” in the pcDNA3.1 vector. (RLuc8 was a gift from Sam Gambhir, Stanford.) cDNAs encoding the split fragments of Nano luciferase (Promega), LgBit (residues 1–158) or HiBit (residues 159–169: VSGWRLFKKIS), were fused in frame to the C-terminus of mGlu8 following the linker “GSPPARAT” in the pcDNA3.1 vector. The following G protein constructs were also used: Gαi-mVenus with the mVenus inserted at position 91, untagged Gβ1, and untagged Gγ2. The integrity of all the constructs was confirmed with sequencing analysis. Cultured Human Embryonic Kidney 293T (HEK293T) cells were transfected with a constant amount of plasmid cDNA using polyethylenimine (Polysciences Inc) in a 1:2 ratio in 10-cm dishes. The ratio of transfected plasmids was optimized to maximize the luminescence of the complemented donor as well as the dynamic range of the BRET response to DL-AP4. For CODA-RET experiments on mGlu7 homodimers, the ratio of mGlu7-L1, mGlu7-L2, Gαi-mVenus, Gβ1, and Gγ2 was 4:4:2:1:1 (for a 10-cm dish, 4, 4, 2, 1, and 1 μg, respectively). For CODA-RET experiments on mGlu7/8 heterodimers, the ratio of mGlu8-L1, mGlu7-L2, Gαi-mVenus, Gβ1, and Gγ2 was 8:4:2:1:1 (for a 10-cm dish, 8, 4, 2, 1, and 1 μg, respectively). For CODA-RET experiments on mGlu8 homodimers, the ratio of mGlu8-LgBit, mGlu8-HiBit, Gαi-mVenus, Gβ1, and Gγ2 was 4:4:6:1:1 (for a 10-cm dish, 4, 4, 6, 1, and 1 μg, respectively). Cells were maintained in culture with DMEM supplemented with 10% FBS. Experiments were performed 48 h after transfection.

      CODA-RET assay

      Cells were harvested, washed twice, and resuspended in 1× Dulbecco′s Phosphate Buffered Saline. Approximately 300,000 cells per well were distributed in 96-well plates and stimulated by the indicated drugs dissolved in prewarmed 1× Dulbecco′s Phosphate Buffered Saline for 15 min at 37 °C. A concentration of 5 μM coelenterazine H (the substrate used for both complemented RLuc8 and NanoLuc) was added to each well (Dalton Pharma Services). Two minutes after the addition of coelenterazine H, the fluorescence and luminescence were quantified (Pherastar, BMG Labtech) and the BRET signal was determined by calculating the ratio of the emission of mVenus (535 nm) over the emission of RLuc8 or NanoLuc (475 nm).

      Electrophysiology

      Animals were group housed with food and water available ad libitum. Animals were kept under a 12-h light/dark cycle with lights on from 6:00 AM to 6:00 PM, and slices were prepared during the light phase. All of the experimental procedures were approved by the Vanderbilt University Animal Care and Use committee and followed the guidelines set forth by the Guide for the Care and Use of Laboratory Animals. Six- to eight- week-old male C57BL6/J mice (Jackson Laboratories) were anesthetized with isofluorane, and the brains were removed and submerged in ice-cold cutting solution (in mM: 230 sucrose, 2.5 KCl, 8 MgSO4, 0.5 CaCl2, 1.25 NaH2PO4, 10 D-glucose, 26 NaHCO3). Coronal slices containing the hippocampus were cut at 400 μm using a Compresstome (Precisionary Instruments). Slices were transferred to a holding chamber containing NMDG-HEPES recovery solution (in mM: 93 NMDG, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 D-glucose, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 10 MgSO4, 0.5 CaCl2, pH 7.3–7.4, 305 mOsm) for 10 min at 32 °C. Slices were then transferred to a room temperature holding chamber for at least 1 h containing artificial cerebrospinal fluid (ACSF) (in mM: 126 NaCl, 1.25 NaH2PO4, 2.5 KCl, 10 D-glucose, 26 NaHCO3, 2 CaCl2, 1 MgSO4) supplemented with 600 μM sodium ascorbate for slice viability. All buffers were continuously bubbled with 95% O2/5% CO2. Subsequently, slices were transferred to a 32 °C submersion recording chamber where they were perfused with ACSF at a rate of 2 ml/min. Paired-pulse fEPSPs were recorded from the stratum radiatum of CA1 and evoked by electrical stimulation (100 μs duration, every 20 s) through a concentric bipolar stimulating electrode placed near the CA3-CA1 border. Input–output curves were generated for each slice, and the stimulation intensity was adjusted to 40 to 50% of the maximum response. After 10 min of baseline recordings, mGlu7 NAMs or vehicle were bath applied for 10 to 20 min.
      LTP was induced by either HFS or TBS. HFS comprised two trains of 100 Hz stimulation (1 s duration, 20 s intertrain interval). TBS consisted of four trains of nine bursts, with each burst containing four pulses at 100 Hz and interburst interval of 100 ms and intertrain interval of 10 s. Data were digitized using a Multiclamp 700B, Digidata 1322A, and pClamp 10 software (Molecular Devices) and were analyzed offline using Clampfit 10.2 (Molecular Devices). For analysis, the slopes from three sequential sweeps were averaged. To test the effects of various treatments on the slope, all slopes were normalized to the averaged slopes during the predrug period (10-min baseline) and were presented as the percent of baseline. All drugs were diluted in ACSF and bath applied.

      Data availability

      All data are contained within the manuscript and are available upon request from Colleen M. Niswender ( [email protected] ) and Jonathan A. Javitch ( [email protected] ).

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank William K. Warren, Jr, and the William K. Warren Foundation who funded the William K. Warren Jr Chair in Medicine (to C. W. L.), as well as endowment of the Warren Center for Neuroscience Drug Discovery at Vanderbilt.

      Author contributions

      X. L., Z. X., C. M. N., and J. A. J. conceptualization; X. L., N. M. F., S. D., R. K. S., C. W. R., J. J. K., and C. W. L. methodology; X. L., N. M. F., S. D., and R. K. S. formal analysis; X. L., N. M. F., S. D., and R. K. S. investigation; X. L., N. M. F., S. D., R. K. S., C. W. R., J. J. K., C. W. L., and Z. X. writing – review and editing; N. M. F., S. D., R. K. S., and W. B. A. validation; C. W. R., J. J. K., and C. W. L. resources; W. B. A., C. M. N., and J. A. J. writing – original draft; W. B. A., C. M. N., and J. A. J. visualization; Z. X., C. M. N., and J. A. J. supervision; C. M. N. and J. A. J. funding acquisition.

      Funding and additional information

      This work was supported by NIH, United States grants MH113543 (C. W. L./C. M. N.), MH124671 (C. W. L./C. M. N.), NS113614 (J. A. J.), and MH054137 (J. A. J.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by the Department of Defense, United States Congressionally Directed Medical Research Program grant W81XWH-17-1-0266 (C. M. N.), the Hope for Depression Research Foundation, United States (J. A. J.), and Miriam’s Magical Memorial Mission (J. A. J.).

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