Activation of NR2A-containing NMDA receptors is not obligatory for NMDA receptor-dependent long-term potentiation

Abstract

Activation of NMDA receptors (NMDARs) within the CNS represents a major signal for persistent alterations in glutamatergic signaling, such as long-term potentiation (LTP) and long-term depression. NMDARs are composed of a combination of NR1 and NR2 subunits, with distinct NR2 subunits imparting distinct characteristics on the receptor. One particular NR2 subunit, NR2A (NRepsilon1), has been proposed to play an integral role in LTP induction in the hippocampus and cortex. Here, we report studies investigating the role of NR2A in LTP induction in the dorsolateral bed nucleus of the stria terminalis (dlBNST). The putative NR2A-specific inhibitor NVP-AAM077 (AAM077) has been used previously to demonstrate the dependence of cortical and hippocampal LTP on NMDARs containing NR2A subunits. We report here the same sensitivity of LTP to pretreatment with AAM077 (0.4 microm) in the dlBNST. However, inconsistent with the conclusion that LTP in the dlBNST is NR2A dependent, we see intact LTP in the dlBNST of NR2A knock-out mice. Because we also see blockade of this dlBNST LTP in NR2A knock-out mice after pretreatment with AAM077, we conclude that the antagonist is targeting non-NR2A subunit-containing receptors. Using a variety of cultured cell types, we find that AAM077 (0.4 microm) can attenuate transmission of NR2B subunit-containing NMDARs when preapplied rather than coapplied with an agonist. Therefore, we conclude that NR2A is not obligatory for the induction of LTP in the dlBNST. Furthermore, our data demonstrate that care must be exercised in the interpretation of data generated with AAM077 when the compound is applied before an agonist.

Figure 1.

NMDAR-dependent LTP in the dlBNST does not require NR2A subunit-containing receptors. A, Averaged time course of synaptic field potentials after high-frequency stimulation (2 1 s trains, 100 Hz) in coronal slices containing the dlBNST from adult C57BL/6J mice. Pretreatment with AAM077 (0.4 μm) for 30 min significantly attenuated LTP (open circles) compared with interleaved controls (filled squares) (p < 0.05). B, LTP in the dlBNST was unaffected in NR2A knock-outs (open squares) compared with wild-type (WT) littermates (filled triangles) (p = 0.28). LTP in NR2A knock-outs was blocked by pretreatment with AAM077 (0.4μm; open circles) (p < 0.05).

Figure 2.

Attenuation of NMDA-induced currents by NR2-specific inhibitors in HEK cells expressing recombinant NMDARs. A, Representative traces of NMDA-induced currents in HEK cells expressing NR1–1a/NR2A (A₁) and NR1–1a/NR2B (A₂) under control conditions, after preapplication of AAM077 (0.4 μm), after preapplication of ifenprodil (3 μm), and after washout. B, Averaged amount of attenuation of NMDA-induced currents by AAM077 (0.4μ m) in HEK cells expressing NR1–1a/NR2A (left) and NR1–1a/NR2B (right). C, Averaged amount of attenuation of NMDA-induced currents by ifenprodil (3 μm) in HEK cells expressing NR1–1a/NR2A (left) and NR1–1a/NR2B (right). Values are expressed as the ratio of the response in the presence of NMDA plus antagonist compared with the response in the presence of NMDA alone. The numbers of averaged cells are indicated in parentheses above the bars. sim, Simultaneous application of antagonist and NMDA; pre, preapplication of antagonist 30 s before NMDA; pk, peak current (measured at the time point at which control currents reach maximum amplitude); ss, steady-state current.

Figure 3.

Attenuation of NMDA-mediated current by NR2-specific inhibitors in dispersed cultured hippocampal neurons (3–4 d in vitro). A, Representative traces of NMDA-induced currents under control conditions, after preapplication of AAM077 (0.4 μm), after preapplication of ifenprodil (3 μm), and after washout. B, Averaged amount of attenuation of NMDA-induced currents by AAM077 (0.4 μm). C, Averaged amount of attenuation of NMDA-induced currents by ifenprodil (3 μm). The numbers of averaged neurons are indicated in parentheses above the bars. Data were obtained from three separate culture dishes from two separate litters for each condition. sim, Simultaneous application of antagonist and NMDA; pre, preapplication of antagonist 30 s before NMDA; pk, peak current (measured at the time point at which control currents reach maximum amplitude); ss, steady-state current.

Figure 4.

Attenuation of NMDAR-mediated, but not AMPAR-mediated, transmission by NR2-specific inhibitors in autaptic cell cultures from the hippocampus of NR2A knock-out mice. A, Representative traces of synaptically evoked NMDAR-mediated EPSCs under control conditions in the presence of AAM077 (0.4 μm), in the presence of ifenprodil (3 μm), and after washout. B, Averaged amount of attenuation of synaptically evoked NMDAR-mediated EPSCs by ifenprodil (3 μm; gray bar) and AAM077 (0.4 μm; black bar). C, Representative traces of synaptically evoked AMPA-mediated EPSCs under control conditions, in the presence of AAM077(0.4μm), in the presence of ifenprodil (3 μm), and after washout. D, Averaged amount of attenuation of synaptically evoked AMPA-mediated EPSCs (peak) and paired-pulse ratios (ppr) by AAM077 (0.4 μm; closed bars) and ifenprodil (3 μm; open bars). The numbers of averaged neurons are indicated in parentheses above the bars. Data were obtained from a single culture for each condition.