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. 2018 Dec 12;142(1):80–92. doi: 10.1093/brain/awy304

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© The Author(s) (2018). Published by Oxford University Press on behalf of the Guarantors of Brain.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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No compensation by GluN2B. GluN2B-mediated currents do not increase to compensate for GluN2A deficiency in cortical neurons from a Grin2a knock-out rat. (A) Western blot and quantification confirming the absence of GluN2A expression in Grin2a^−/− neurons, and an intermediate expression level in Grin2a^+/− neurons at 15 DIV. Tukey’s test reveals a significant difference between Grin2a^+/+ versus Grin2a^+/− (P = 0.0196) and versus Grin2a^−/− (P = 0.0014). Grin2a^+/+: n = 6; Grin2a^+/−: n = 5; Grin2a^−/−: n = 5. (B) Western blot and quantification confirming no changes in GluN2B expression in either Grin2a^+/− or Grin2^−/− neurons compared to Grin2a^+/+ neurons at 15 DIV. Grin2a^+/+, n = 6; Grin2a^+/−: n = 5; Grin2a^−/−: n = 5. (C) NMDA (150 µM) evoked currents were measured in cortical neurons of the indicated genotypes and periods of culture. Currents were calculated and normalized to cell capacitance to give a value for the current density within the neuron. Two-way ANOVA reports a significant developmental stage effect (P < 0.0001) and a significant genotype effect (P = 0.013) as well as a significant interaction between the two (P = 0.0059). Sidak’s post hoc test reveals a significant difference between Grin2a^+/+ versus Grin2a^+/− (P = 0.0007) and versus Grin2a^−/− (P = 0.0006). Grin2a^+/+: n = 38 (7–8 DIV), 38 (15–16 DIV) cells, eight animals; Grin2a^+/−: n = 40 (7–8 DIV), 35 (15–16 DIV) cells, nine animals; Grin2a^−/−: n = 48 (7–8 DIV), 31 (15–16 DIV) cells, 10 animals. (D) NMDA (150 µM) evoked currents were measured in cortical neurons of the indicated genotypes and periods of culture before and after the application of the GluN2B-selective antagonist ifenprodil (3 µM). The ifenprodil-sensitive current was calculated and normalized to cell capacitance. Two-way ANOVA reports a significant developmental stage effect (P < 0.0001) but no significant genotype effect (P = 0.880) nor a significant interaction between the two (P = 0.154). Grin2a^+/+: n = 13 (7–8 DIV), 13 (15–16 DIV) cells, four animals; Grin2a^+/−: n = 13 (7–8 DIV), 11 (15–16 DIV) cells, five animals; Grin2a^−/−: n = 17 (7–8 DIV), 16 (15–16 DIV) cells, five animals. (E) At 15–16 DIV the percentage inhibition of NMDA (150 µM) evoked currents by ifenprodil (3 µM) was significantly greater (Tukey’s test) in Grin2a^−/− neurons compared to Grin2a^+/+ neurons (P < 0.0001) and Grin2a^+/− neurons (P = 0.0038). DIV = day in vitro.