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Role of NMDA Receptor Subtypes in Different Forms of NMDA-dependent Synaptic Plasticity

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Role of NMDA Receptor Subtypes in Different Forms of NMDA-dependent Synaptic Plasticity

Rui Li et al. BMC Neurosci.

Abstract

Background: The involvement of different NMDA receptor (NMDAR) subunits has been implicated in several forms of synaptic plasticity. However, it is still controversial to what extent the involvement is specific, and little is known about the role of NMDAR subunits in certain "non-conventional" forms of plasticity. In this study we used subunit-specific blockers to test the roles of NR2A- and NR2B-containing NMDARs in a type of chemical long-term depression (LTD) induced by brief bath application of the NMDAR agonist NMDA to hippocampal slices from 12-18 days old rats. For comparison, we also examined other forms of plasticity, including a "slow LTD" induced by 0.1 Hz stimulation under low Mg2+ conditions as well as long-term potentiation (LTP).

Results: A blocker of NR2A-containing NMDARs, NVP-AAM077 (NVP), substantially reduced the two forms of studied depression whereas blockers of NR2B-containing NMDARs, Ro25-6981 (Ro) or Ifenprodil (Ife), had no significant effect on them. LTP appeared to be more sensitive as it was fully blocked by NVP and partially blocked by Ro or Ife. However, the blocking effects of NVP could be counteracted by general amplification of NMDA responses by lowering Mg2+ concentration in the perfusion solution. Applying NVP or Ro/Ife on isolated NMDA-EPSPs recorded in low Mg2+ solution reduced responses to about 70% and 20% of initial size, respectively, whereas coapplication of both blockers almost completely abolished the responses. Additionally, NMDA application caused depotentiation of a pathway with prior tetanus-induced LTP, and NVP but not Ro/Ife substantially prevented that depotentiation as well as the chemical LTD of the control pathway. A second tetanus on the LTP pathway induced repotentiation which was fully blocked by NVP but partially blocked by Ro/Ife.

Conclusion: All of these results on hippocampal slices from young rats can be explained by a simple model, in which NR2A subunits dominate over NR2B subunits with respect to both plasticity and NMDAR-mediated responses. The model suggests that Ca2+ influx into the postsynaptic spine via different subtypes of NMDARs makes up a "final common pathway", controlling synaptic plasticity by its magnitude and temporal pattern regardless of the source.

Figures

Figure 1
Figure 1
Roles of NR2B and NR2A subunits in NMDA-induced LTD. (A) NMDA application (30 μM, 4 min) under normal conditions causes initial extinction of EPSPs, followed by recovery and stabilization (n = 15). (B) NR2B inhibitors, Ro25-6981 (0.5 μM) or Ifenprodil (3 μM), do not affect NMDA-induced plasticity (n = 8+4 = 12). (C) In presence of NR2A inhibitor, NVP-AAM077 (0.4 μM), NMDA effects are different compared with control situation, with a shorter recovery phase and much less depression (n = 10). (D) Bar diagram reveals LTD at 60 min after NMDA application as percentage of the initial baseline under the different experimental conditions in A-C. Data are given as mean ± S.E.M. Black bars in A-C indicate the duration of drug treatment. Values are shown averaged for 2 min periods. Inserts illustrate EPSP-traces taken at the indicated time points (a, b). Calibrations: 0.5 mV, 10 ms.
Figure 2
Figure 2
Relative contributions of NR2B and NR2A subunits to isolated NMDA-EPSPs. (A) Isolated NMDA-EPSPs obtained in a low Mg2+ solution (0.1 mM; n = 5). (i) After recording baseline responses, defining the 100% level (1), applying NR2A inhibitor NVP-AAM077 (0.4 μM) leads to a substantial reduction of the isolated NMDA response (2). Adding NR2B inhibitor Ro25-6981 (0.5 μM) further depresses the responses down to near zero (3). Subsequent perfusion with AP5 (50 μM) fully blocks the synaptic responses and values obtained in this solution are taken as zero level (4). (ii) Traces 1–3 plotted together after subtraction of the zero level. (iii) Bar diagram quantifying the reductions of NMDA-EPSPs after sequentially adding the two subunit-specific blockers, NVP-AAM077 and Ro25-6981. (B) Similar plots for another set of experiments where the two blockers were applied in a different order (n = 6). The bar diagrams show data as mean ± S.E.M. Calibrations: 0.1 mV, 20 ms.
Figure 3
Figure 3
Roles of NR2B and NR2A subunits in stimulus-induced LTD. (A) A "slow LTD" is induced by 0.1 Hz test rate stimulation for 90 min in a low Mg2+ solution. The experiment consists of preinduction baseline level in presence of a high concentration of AP5 (50 μM), induction period in AP5-free solution, and established LTD after reintroduction of high AP5. AMPA component (black symbols) and NMDA component (gray symbols) are plotted as functions of time, each point indicating the average in 2 min (n = 8 experiments). (B) A similar result is obtained in the same type of experiment but treated with Ro25-6981 (0.5 μM; n = 8) during the induction period. (C) The "slow LTD" is substantially blocked when NVP-AAM077 (0.4 μM; n = 7) is present under induction in AP5-free solution. (D) Bar diagram shows the "slow LTD" at 30 min after reintroducing AP5, plotted as percentage of the preinduction level under the three different experimental situations in A-C. Data are mean ± S.E.M. Black bars in A-C indicate the duration of drug treatment. Inserts show the EPSP-traces taken at the indicated time points (a-d) and the timing of the measurements for AMPA and NMDA components (0–1.5 ms after fiber volley and at 35–45 ms, respectively; see bars below traces). Calibrations: 0.2 mV, 20 ms.
Figure 4
Figure 4
Roles of NR2B and NR2A subunits in LTP. (A) LTP induced by three successive tetani (100 Hz, 100 impulses each) in normal solution. The EPSP is potentiated to a near doubling of the initial response. Test pathway (black symbols) and control pathway (gray symbols) are plotted as functions of time, each point indicating the average in 1 min (n = 10 experiments). (B) LTP is partially blocked by Ro25-6981 (0.5 μM; n = 2)/Ifenprodil (3 μM; n = 4) (total n = 6). (C) LTP is fully prevented when NVP-AAM077 (0.4 μM) is present (n = 5). (D) Bar diagram summarizes the data in A-C with mean ± S.E.M. LTP is measured at 60 min after tetanization relative to the initial baseline. Arrows in A-C indicate the tetani. Black bars indicate the duration of drug application. Inserts illustrate EPSP-traces taken at the indicated time points (a, b). Calibrations: 0.5 mV, 20 ms.
Figure 5
Figure 5
Roles of NR2B and NR2A subunits in depotentiation and repotentiation after LTP. (A) LTP is induced by three successive tetani (100 Hz, 100 impulses each) leading to a near doubling of the EPSP. Subsequent application of NMDA (30 μM, 4 min) leads to persistent depression of the control pathway (gray symbols) and depotentiation of the test pathway (black symbols). Secondary tetanization of the test pathway causes a repotentiation, lifting the test responses back to a potentiated level (n = 12). (B) and (C) show similar experiments but treated with Ro25-6981 (0.5 μM; n = 4)/Ifenprodil (3 μM; n = 2) (total n = 6) or NVP-AAM077 (0.4 μM; n = 5) shortly after LTP induction. Both depotentiation and repotentiation are preferentially attenuated by NVP-AAM077 as compared to Ro25-6981/Ifenprodil. Arrows indicate the tetani. Black bars indicate the duration of drug application.
Figure 6
Figure 6
Summary of depotentiation and repotentiation effects. (A) Key, defining the measurements used. The experiment model indicates the three types of plasticity, LTP, depotentiation, and repotentiation, subjected to measurements at times (1), (2) and (3), respectively. Additionally, there are three types of experiment with different drug treatment: normal (n = 12), NR2B block (by Ro25-6981/Ifenprodil, n = 6) and NR2A block (by NVP-AAM077, n = 5), denoted a-c and corresponding to panels A-C in Fig. 5 (same primary data used). Since drugs were applied after LTP induction only depotentiation and repotentiation were subjected to the specific subunit blockers. (B) Values of field EPSPs are estimated as test responses in percentage of initial baseline under the three different experimental conditions (a-c) and time points (1–3). (C) Same as B but values are calculated as the ratio between test and control responses for the three types of experiment. Data are expressed as mean ± S.E.M.
Figure 7
Figure 7
Facilitated induction of plasticity overrides the blockade by NVP-AAM077. (A) Blocking effect of NVP-AAM077 on NMDA-induced LTD (see Fig. 1C) is overcome by lowering Mg2+ concentration (0.1 instead of 1.3 mM; n = 6). (B) Bar diagram shows NMDA-induced LTD in different Mg2+ solutions. Dashed bar is control data from Fig. 1D. (C) In a "slow LTD" experiment, similar to that in Fig. 3C, lowering the concentration of Mg2+ in the perfusion solution (0.01 instead of 0.1 mM) helps with the induction of "slow LTD" in presence of NVP-AAM077 (0.4 μM; n = 7). (D) Bar diagram reveals the "slow LTD" induced in different Mg2+ solutions. Dashed bar is control data from Fig. 3D. (E) Similarly, using low Mg2+ solution (0.1 instead of 1.3 mM) in LTP experiments (compare Fig. 4C) enables a small potentiation of EPSPs under inhibition of NR2A subunits by NVP-AAM077 (n = 7). (F) Bar diagram shows LTP induced in different Mg2+ solutions. Dashed bar is control data from Fig. 4D. Data are shown as mean ± SEM. Inserts show the EPSP-traces taken at the indicated time points (a-d). Calibrations: 0.5 mV (A, E), 0.2 mV (C), 20 ms.

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