NMDA Glun2c/2D Receptors Contribute to Synaptic Regulation
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Chen et al. Mol Brain (2021) 14:60 https://doi.org/10.1186/s13041-021-00744-3 RESEARCH Open Access NMDA GluN2C/2D receptors contribute to synaptic regulation and plasticity in the anterior cingulate cortex of adult mice Qi‑Yu Chen1,2, Xu‑Hui Li1,2,3, Jing‑Shan Lu1,2, Yinglu Liu1,3, Jung‑Hyun Alex Lee3, Yu‑Xin Chen1, Wantong Shi1, Kexin Fan1 and Min Zhuo1,2,3* Abstract Introduction: N‑Methyl‑D‑aspartate receptors (NMDARs) play a critical role in diferent forms of plasticity in the cen‑ tral nervous system. NMDARs are always assembled in tetrameric form, in which two GluN1 subunits and two GluN2 and/or GluN3 subunits combine together. Previous studies focused mainly on the hippocampus. The anterior cingu‑ late cortex (ACC) is a key cortical region for sensory and emotional functions. NMDAR GluN2A and GluN2B subunits have been previously investigated, however much less is known about the GluN2C/2D subunits. Results: In the present study, we found that the GluN2C/2D subunits are expressed in the pyramidal cells of ACC of adult mice. Application of a selective antagonist of GluN2C/2D, (2R*,3S*)‑1‑(9‑bromophenanthrene‑3‑carbonyl) piperazine‑2,3‑dicarboxylic acid (UBP145), signifcantly reduced NMDAR‑mediated currents, while synaptically evoked EPSCs were not afected. UBP145 afected neither the postsynaptic long‑term potentiation (post‑LTP) nor the presynaptic LTP (pre‑LTP). Furthermore, the long‑term depression (LTD) was also not afected by UBP145. Finally, both UBP145 decreased the frequency of the miniature EPSCs (mEPSCs) while the amplitude remained intact, suggesting that the GluN2C/2D may be involved in presynaptic regulation of spontaneous glutamate release. Conclusions: Our results provide direct evidence that the GluN2C/2D contributes to evoked NMDAR mediated cur‑ rents and mEPSCs in the ACC, which may have signifcant physiological implications. Keywords: NMDAR, GluN2C/2D, ACC , LTP, LTD Introduction two GluN1 subunits and two GluN2 and/or GluN3 sub- As a key glutamate-gated ion channel in the central nerv- units combine together as di-heteromeric or tri-hetero- ous system, N-Methyl-d-aspartate receptors (NMDARs) meric receptors. Te subunit composition varies during attract a lot of attention because of their known roles in neurodevelopment, and determines channel properties synaptic plasticity [1, 2]. Subunits of NMDARs are clas- of NMDARs [3]. Among all the subunits, NMDARs with sifed in three main families which are identifed accord- GluN2C/2D subunits display lower sensitivity to Mg2+ ing to the homogeneity: GluN1, GluN2A/2B/2C/2D and ions and slower deactivation kinetics compared with GluN3A/3B. Structural studies have already reported that GluN2A/2B containing NMDARs. GluN2C/2D-con- NMDARs are assembled in tetrameric form, in which taning NMDARs could allow selective modifcation of circuit function in regions expressing GluN2C/2D *Correspondence: min.zhuo@utoronto.ca subunits[4–7]. Te expression of GluN2C/2D has been 1 Center for Neuron and Disease, Frontier Institutes of Science reported in adult cerebral cortices [8–13]. GluN2D subu- and Technology, Xi’an Jiaotong University, Xi’an, China nits were reported to play roles in synaptic transmission Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chen et al. Mol Brain (2021) 14:60 Page 2 of 13 of hippocampal neurons[14, 15]. Te activation of were purchased from Tocris Cookson (Bristol, UK), PTX NMDARs by glutamate spillover was prevented by the was bought from Sigma-Aldrich (St Louis, MO, USA). GluN2D-selective antagonists. Drugs were prepared as stock solutions for frozen ali- In the anterior cingulate cortex (ACC), a critical cor- quots at -20℃. All drugs were diluted from the stock tical region involved in chronic pain and emotions, the solution to the fnal desired concentration in the ACSF roles of NMDAR subunits in synaptic plasticity have before being applied to the perfusion solution. been investigated [2, 16, 17]. Application of the NMDAR antagonist AP-5 blocked both the NMDAR- mediated Brain slices preparation EPSCs and the induction of the postsynaptic form of Coronal brain slices (300 μm) of ACC were prepared long-term potentiation (post-LTP). Both application of using standard methods. Briefy, mice were deeply anes- the GluN2A antagonist PEAQX and of GluN2B antago- thetized with 5% isofurane and sacrifced by decapita- nist ifenprodil or Ro 25-6981 blocked the NMDAR- tion. Te whole brain was removed quickly from the skull mediated EPSCs, which indicates NMDAR containing and submerged in the oxygenated (95% O 2 and 5% CO 2) GluN2A or GluN2B subunits contribute to most of the ice-cold artifcial cerebrospinal fuid (ACSF) contain- NMDAR currents [18]. Te application of antagonist ing (in mM) 124 NaCl, 2.5 KCl, 2 MgSO4, 1 NaH2PO4, 2 also reduced, or completely abolished (co-application CaCl2, 25 NaHCO3, and 10 D-glucose. Te whole brain of both inhibitors) the post-LTP. GluN2A and GluN2B tissue was cooled for short time before the tissue con- were also proved to be required for long-tern depres- taining the target region was isolated and glued onto the sion (LTD) in the ACC [19]. Aside from the focus on vibratome (VT1200S Vibratome, Leica, Germany). Slices the GluN2A/2B, genes encoding GluN2C and GluN2D were incubated in a submerged recovery chamber at have also been detected by in-situ hybridization in the room temperature for one hour. Te ACSF was continu- ACC of mice (http://mouse .brain -map.org/exper iment / ously balanced with a mixture of 95% O2 and 5% CO2. show/75888 748, http://mouse .brain -map.org/exper iment /show/75551 479). GluN2C and GluN2D were detected in Western blot the ACC of humans by western blot [20], but less stud- Brain regions were homogenized in 10 mM Tris (pH ies have been carried out on the function of GluN2C/2D 7.4), 2 mM EDTA, and 1% SDS, 1× protease inhibitor containing NMDARs in the ACC of mice. cocktail. Lysates were centrifuged at 14,000 rpm, 4 °C Terefore, in this study, we frst detected the expres- for 20 min, and the supernatants were used for protein sion of GluN2C/2D in the ACC of adult mice by Western analysis and Western blotting. Equal amounts (40–50 μg) blot. Ten we performed whole-cell patch-clamp record- of protein from tissue lysates were separated by SDS- ings from the brain slices of the ACC in adult mice. By PAGE and transferred to polyvinylidene difuoride mem- using a selective antagonist of GluN2C/2D, (2R*,3S*)- brane followed by blocking with 5% skim milk for 1 h at 1-(9-bromophenanthrene-3-carbonyl) piperazine-2,3-di- room temperature. Blots were probed with anti-GluN2A carboxylic acid (UBP145), we found that the GluN2C/2D (1:1000; Millipore, US), anti-GluN2B (1:500; Millipore, modulates synaptic transmission in a presynaptic pat- US), anti-GluN2C (1:500; Millipore, US) or anti-GluN2D tern, and they partially composed the NMDAR-mediated (1:500; Millipore, US) polyclonal antibodies overnight at EPSCs in pyramidal neurons of the ACC. Our results 4 °C. For tubulin blotting as a control, a monoclonal anti- indicate that GluN2C/2D may also be involved in the body (1:4000; Sigma, US) was used. Te membranes were functions of NMDARs in the ACC. incubated with a horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse IgG (1:5000; Millipore, US) Material and methods for 1 h at room temperature, and the bands were visual- Animals ized by an ECL system (GE Healthcare, US). Exposure Adult male C57BL/6 mice (7–9 weeks old) were used. All time varied for each primary antibody to ensure that the animals were housed under a 12 h light/dark cycle with signals were in the linear range. Signals were quantifed food and water provided ad libitum. Experiments were using ImageJ software. conducted under the protocol approved by the Ethics Committee of Xi’an Jiaotong University and the Univer- Whole‑cell patch‑clamp recording sity of Toronto. Whole cell recordings were performed in a recording chamber on the stage of an Olympus BX51 microscope Drugs with infrared diferential interference contrast (DIC) Te chemical and drugs used in this study were as fol- optics for visualization. EPSCs were recorded from layer lows: AP-5, CNQX and Ro 25-6981 purchased from Hel- II/III neurons with an Axon 200B amplifer (Molecular loBio (Princeton, NJ, USA), PPDA, UBP145 and PEAQX Devices), and the stimulations were evoked in layer V of Chen et al. Mol Brain (2021) 14:60 Page 3 of 13 the ACC by a bipolar tungsten stimulating electrode. Te recording pipettes (3–5 MΩ) were flled with the solution containing (in mM) 145 K-gluconate, 5 NaCl, 1 MgCl2, 0.2 EGTA, 10 HEPES, 2 Mg-ATP, and 0.1 Na3-GTP, which adjusted to pH 7.3 with KOH and had osmolality of 300 mOsmol.