Distinct roles of NR2A and NR2B cytoplasmic tails in long-term potentiation

Abstract

NMDA receptors (NMDARs) are critical mediators of activity-dependent synaptic plasticity, but the differential roles of NR2A- versus NR2B-containing NMDARs have been controversial. Here, we investigate the roles of NR2A and NR2B in long-term potentiation (LTP) in organotypic hippocampal slice cultures using RNA interference (RNAi) and overexpression, to complement pharmacological approaches. In young slices, when NR2B is the predominant subunit expressed, LTP is blocked by the NR2B-selective antagonist Ro25-6981 [R-(R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propranol]. As slices mature and NR2A expression rises, activation of NR2B receptors became no longer necessary for LTP induction. LTP was blocked, however, by RNAi knockdown of NR2B, and this was rescued by coexpression of an RNAi-resistant NR2B (NR2B*) cDNA. Interestingly, a chimeric NR2B subunit in which the C-terminal cytoplasmic tail was replaced by that of NR2A failed to rescue LTP, whereas the reverse chimera, NR2A channel with NR2B tail, was able to restore LTP. Thus, expression of NR2B with its intact cytoplasmic tail is required for LTP induction, at an age when channel activity of NR2B-NMDARs is not required for LTP. Overexpression of wild-type NR2A failed to rescue LTP in neurons transfected with the NR2B-RNAi construct, despite restoring NMDA-EPSC amplitude to a similar level as NR2B*. Surprisingly, an NR2A construct lacking its entire C-terminal cytoplasmic tail regained its ability to restore LTP. Together, these data suggest that the NR2B subunit plays a critical role for LTP, presumably by recruiting relevant molecules important for LTP via its cytoplasmic tail. In contrast, NR2A is not essential for LTP, and its cytoplasmic tail seems to carry inhibitory factors for LTP.

Figure 1.

Increasing NR2A expression and falling NR2B contribution to NMDA–EPSC during development of hippocampal slice cultures. A, Immunoblot of hippocampal slices from P7 rats cultured for the indicated number of days in vitro (left). Graph showing intensity at a given DIV normalized to intensity at DIV 0 (right). B, Sample traces and histograms showing the effects of 5 μm Ro25-6981 (left), 50 nm NVP-AAM077 (middle), and 5 μm Ro25-6981 followed by 50 nm NVP-AAM077 plus 5 μm Ro25-6981 (right) on the NMDA–EPSC amplitude. Left and middle show traces from DIV 10–14 neurons taken before (dashed line) and after (solid line) drug application in both absolute value and scaled for comparison of kinetics. Right shows traces without drug (largest trace), after 5 μm Ro25-6981 (middle trace), and after 5 μm Ro25-6981 plus 50 nm NVP-AAM077 (smallest trace). Histograms show the percentage of the NMDA–EPSC amplitude that is blocked by the drugs at DIV 6–8, DIV 10–14, and DIV 18–21 (n = 12, 18, and 6, respectively, for Ro25-6981; n = 5, 5, and 6, respectively, for NVP-AAM077; n = 5 for Ro25-6981 plus NVP-AAM077.) Data are presented as mean ± SEM. *p < 0.05, Student's t test.

Figure 2.

Effect of Ro25-6981 on LTP in cultured hippocampal slices over development. A, B, Time course of LTP in control conditions (open circles) and in the presence of 5 μm Ro25-6981 (filled circles) at DIV 6–8 (A; n = 10 for control; n = 6 for Ro25-6981; p < 0.05) and DIV 11–14 (B; n = 12 for control; n = 9 for Ro25-6981; p = 0.86) (Wilcoxon's test at 20–30 min) Insets show example averaged EPSCs taken before and 30 min after pairing for control cells (left, open circle) and cells in the presence of Ro25-6981 (right, filled circle).

Figure 3.

Effect of altering NR2B expression on LTP. A, Histograms showing the average ratio of the NMDA–EPSC (n = 13, 14, and 6) (a), the ratio of the half-width (HW) (n = 13, 14, and 6) (b), and the ratio of the AMPA–EPSC in transfected cells to that of untransfected cells (n = 11, 13, and 10) (c), respectively, for NR2B–RNAi, NR2B* plus NR2B–RNAi, and NR2B). B–D, Time course of LTP for untransfected cells (open circles) and neighboring cells transfected with NR2B–RNAi (n = 10; p < 0.04) (B), NR2B* plus NR2B–RNAi (n = 6; p = 0.7) (C), or NR2B (n = 6; p = 0.9) (D) (Wilcoxon's test at 20–30 min). Insets show example averaged EPSCs taken before and 30 min after pairing. *p < 0.05.

Figure 4.

Effect of altering NR2A expression on LTP. A, Histograms showing the average ratio of the NMDA–EPSC (n = 21, 6, and 6) (a), the ratio of the half-width (HW) (n = 21, 6, and 6) (b), and the ratio of the AMPA–EPSC in transfected cells to that of untransfected cells (n = 13, 13, and 10) (c), respectively, for NR2A–RNAi, NRB RNAi plus NR2A, or NR2A. B–D, Time course of LTP for untransfected cells (open circles) and neighboring cells transfected with NR2A–RNAi (n = 11; p = 0.2) (B), NR2A plus NR2B–RNAi (n = 8; p < 0.007) (C), and NR2A (n = 7; p < 0.05) (D) (Wilcoxon's test at 20–30 min). Insets show example averaged EPSCs taken before and 30 min after pairing. *p < 0.05.

Figure 5.

Effect of the of NR2A and NR2B chimeric constructs on LTP. A, Histograms showing the average ratio of the NMDA–EPSC (n = 11, 5, and 7) (a) and the ratio of the half-widths (HW) (n = 11, 5, and 7) (b) in transfected and untransfected cells for NR2B–RNAi alone and NR2B–RNAi plus NR2B*–NR2Atail or NR2A–NR2Btail, the ratio of the AMPA–EPSC (n = 13 and 7) (c), respectively, for NR2B–RNAi plus NR2B*–NR2Atail or NR2A- NR2Btail. B, C, The time course of LTP in untransfected cells (open circles) and neighboring cells transfected with NR2B–RNAi plus either NR2B*–NR2Atail (n = 7; p < 0.05) (B) or NR2A–NR2Btail (n = 7; p = 0.7) (C) (Wilcoxon's test at 20–30 min).*p < 0.05, **p < 0.01.

Figure 6.

Effect of removing the C-terminal tails of NR2A or NR2B on LTP. A, Histograms showing the average ratio of the NMDA–EPSC (n = 11, 10, and 6) (a) and the ratio of the half-widths (HW) (n = 11, 10, and 6) (b) in transfected and untransfected cells for NR2B–RNAi alone and NR2B–RNAi plus NR2AΔC or NR2B*ΔC, the ratio of the AMPA–EPSC (n = 15 and 9) (c), respectively, for NR2B–RNAi plus NR2AΔC or NR2B*ΔC. B, C, The time course of LTP in untransfected cells (open circles) and neighboring cells transfected with NR2B–RNAi plus either NR2B*ΔC (n = 6; p < 0.05) (B) or NR2AΔC (n = 10; p = 0.1) (C) (Wilcoxon's test at 20–30 min). *p < 0.05, **p < 0.01.

Figure 7.

Effect of mutations in the C-terminal tail of NR2A on LTP. A, Histograms showing the average ratio of the NMDA–EPSC (n = 11, 9, and 8) (a) and the ratio of the half-widths (HW) (n = 11, 9, and 8) (b) in transfected and untransfected cells for NR2B–RNAi alone and NR2B–RNAi plus NR2AΔC₁₂₀₄ or NR2A–S1462A), the ratio of the AMPA–EPSC (n = 9 and 15) (c), respectively, for NR2B–RNAi plus NR2AΔC₁₂₀₄ or NR2A–S1462A. B, C, The time course of LTP in untransfected cells (open circles) and neighboring cells transfected with NR2B–RNAi plus either NR2AΔC₁₂₀₄ (n = 7; p < 0.9) (B) or NR2A–S1462A (n = 7; p < 0.05) (C) (Wilcoxon's test at 20–30 min). *p < 0.05, **p < 0.01.