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. 2018 Sep 7;8:13472. doi: 10.1038/s41598-018-31534-y

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© The Author(s) 2018

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Co-application of glutamate and glycine evokes a Ca²⁺ signal associated with activation of NMDARs through an NR2B independent pathway. (A_1–3) Typical fluorescence images of cortical neurons labelled with Fura-2 (380 nm excitation) (Scale Bar: 100 μm; A₁). Intracellular calcium signals (grey – 27 individual and black – average calcium traces recorded from cultured neurons in A₁) following 2 successive co-applications of glutamate and glycine (2 min; dots; A₂). Calcium traces indicate that there is no detectable difference between the 2 successive Ca²⁺ responses evoked by the co-application of glutamate/glycine. Bar charts comparing the amplitude for Ca²⁺ responses evoked by 2 successive co-application of glutamate/glycine (n_cult = 9; n_cells = 275; A₃). (B_1–3) Intracellular Ca²⁺ signals (black–average calcium traces recorded from 19 neurons in B₁ and 37 neurons in B₂) following 2 successive applications of glutamate/glycine (2 min; dots) in control condition (1^st application) and in presence of the broad-spectrum NMDAR antagonist MK801 (40 μM; B₁) or a specific blocker of the NR2B sub-unit Ifenprodil (2 μM; B₂). The 2^nd Ca²⁺ response induced by the co-application of glutamate/glycine is totally blocked by MK801 (B₁) but not by Ifenprodil (B₂). Bar chart summarizing the significant inhibitory effect of MK801 (n_cult = 8; n_cells = 271) and the lack of effect of Ifenprodil (n_cult = 7; n_cells = 299) on Ca²⁺ responses evoked by the co-application of glutamate/glycine (B₃). Results are presented as means ± SEM (***p < 0.005; paired t-test).