Long-term potentiation in the CA1 hippocampus induced by NR2A subunit-containing NMDA glutamate receptors is mediated by Ras-GRF2/Erk map kinase signaling

Abstract

Background: NMDA-type glutamate receptors (NMDARs) are major contributors to long-term potentiation (LTP), a form of synaptic plasticity implicated in the process of learning and memory. These receptors consist of calcium-permeating NR1 and multiple regulatory NR2 subunits. A majority of studies show that both NR2A and NR2B-containing NMDARs can contribute to LTP, but their unique contributions to this form of synaptic plasticity remain poorly understood.

Methodology/principal findings: In this study, we show that NR2A and NR2B-containing receptors promote LTP differently in the CA1 hippocampus of 1-month old mice, with the NR2A receptors functioning through Ras-GRF2 and its downstream effector, Erk Map kinase, and NR2B receptors functioning independently of these signaling molecules.

Conclusions/significance: This study demonstrates that NR2A-, but not NR2B, containing NMDA receptors induce LTP in pyramidal neurons of the CA1 hippocampus from 1 month old mice through Ras-GRF2 and Erk. This difference add new significance to the observation that the relative levels of these NMDAR subtypes is regulated in neurons, such that NR2A-containing receptors become more prominent late in postnatal development, after sensory experience and synaptic activity.

Figure 1. LTP induced by LFS paring protocol is normal in Ras-GRF2 knock-out mice.

A. Low frequency stimulation (LFS paring) was used to induce LTP in P26-35 wild-type mice (black filled circles; n = 6) and Ras-GRF2 knock-out mice [grf2(−/−), red filled squares; n = 7] and Ras-GRF2 knock-out mice (yellow filled triangles; n = 6) in presence of 100µM APV. Insets, representative EPSC recorded before and after LFS pairing in wild-type (left) and grf2(−/−) (right) slices. Calibration: horizontal, 50 ms; vertical, 50 pA. B Histogram showing no effect of Ras-GRF2 on LTP (p>0.05 vs. WT) but complete inhibition using the NMDAR inhibitor APV.

Figure 2. Effects of NR2B and NR2A-selective antagonists on LFS paring-induced LTP in WT mice.

Hippocampal brain slices from WT mice were preincubated for 30 minutes with either 0.5 µM Ro25-6981(Ro) (black filled circles), 50 ηM NVP -AAM077 (NVP) (red filled squares) or both drugs (green filled triangles). LFS paring-induced LTP was then measured. Neither of the inhibitors had a significant effect on basal synaptic transmission (Suppl. Fig.S1).

Figure 3. Effects of NR2B and NR2A-selective antagonists on LFS paring-induced LTP in Ras-GRF2 knock-out mice.

A. Hippocampal brain slices from Ras-GRF2 knockout mice were perfused throughout the experiments with 0.5 µM of the NR2B selective inhibitor Ro. LFS paring-induced LTP was then measured. B. Hippocampal brain slices from Ras-GRF2 knockout mice were perfused throughout the experiments with 50 ηM of the NR2A selective inhibitor NVP. LFS paring-induced LTP was then measured. Inset: Averaged EPSCs before and after LFS paring in the presence of Ro (left) and NVP (right). Calibration: horizontal, 50 ms; vertical, 50 pA.

Figure 4. LFS paring-induced LTP in double Ras-GRF1/2 knock-out mice.

LFS paring-induced LTP was measured in hippocampal brain slices from double Ras-GRF1/Ras-GRF2 knockout mice in normal (black filled circles; n = 5) and in the presence of 50 ηM NVP (green filled triangles; n = 6) or 0.5 µM Ro (red filled squares; n = 8) ACSF. Inset: Averaged EPSCs before and after LFS paring in control (left) and in the presence of NVP (middle) or Ro (right) ACSF. Calibration: horizontal, 50 ms; vertical, 50 pA.

Figure 5. The effects of MEK and PI3-kinase inhibitors on LFS induced LTP in WT mice.

A LTP was measured in cells in which SL327 (100 µM) was present in recording electrode solution (black filled circles; 0.2% DMSO; n = 7). Tissues were bathed in either 0.5 µM Ro (red filled squares; n = 5) or 50 ηM NVP (green filled triangles; n = 7). B LTP was measured in cells in which LY294002 (Ly) (100 µM) was included in the recording electrode solution (black filled circles; n = 5). These tissues were also bathed in either 0.5µM Ro (red filled squares; n = 5) or 50 ηM NVP (green filled triangles; n = 7). C. Summary graph showing the effects of these drugs on the LTP.

Figure 6. Model of Ras-GRF2 dependent and Ras-GRF independent LTP in hippocampal CA1 pyramidal neurons.

Both NR2A and NR2B-containing NMDARs can produce LTP. However, GRF proteins only make a major contribution to LTP through NR2A-containing receptor signaling through GRF2. Although GRF1 binds to NR2B receptors and contributes to LTD, it does not contribute significantly to NR2B induced LTP. Moreover, like GRF2, Erk mediates only NR2A induced LTP, while PI3K mediates both NR2A and NR2B induced LTP.