Loss of GluN2B-containing NMDA receptors in CA1 hippocampus and cortex impairs long-term depression, reduces dendritic spine density, and disrupts learning

Abstract

NMDA receptors (NMDARs) are key mediators of certain forms of synaptic plasticity and learning. NMDAR complexes are heteromers composed of an obligatory GluN1 subunit and one or more GluN2 (GluN2A-GluN2D) subunits. Different subunits confer distinct physiological and molecular properties to NMDARs, but their contribution to synaptic plasticity and learning in the adult brain remains uncertain. Here, we generated mice lacking GluN2B in pyramidal neurons of cortex and CA1 subregion of hippocampus. We found that hippocampal principal neurons of adult GluN2B mutants had faster decaying NMDAR-mediated EPSCs than nonmutant controls and were insensitive to GluN2B but not NMDAR antagonism. A subsaturating form of hippocampal long-term potentiation (LTP) was impaired in the mutants, whereas a saturating form of LTP was intact. An NMDAR-dependent form of long-term depression (LTD) produced by low-frequency stimulation combined with glutamate transporter inhibition was abolished in the mutants. Additionally, mutants exhibited decreased dendritic spine density in CA1 hippocampal neurons compared with controls. On multiple assays for corticohippocampal-mediated learning and memory (hidden platform Morris water maze, T-maze spontaneous alternation, and pavlovian trace fear conditioning), mutants were impaired. These data further demonstrate the importance of GluN2B for synaptic plasticity in the adult hippocampus and suggest a particularly critical role in LTD, at least the form studied here. The finding that loss of GluN2B was sufficient to cause learning deficits illustrates the contribution of GluN2B-mediated forms of plasticity to memory formation, with implications for elucidating NMDAR-related dysfunction in disease-related cognitive impairment.

Figure 1.

Loss of corticohippocampal GluN2B mRNA expression and protein levels. GluN2B mRNA quantification by in situ hybridization in parasagittal section showed extensive loss of GluN2B mRNA in neocortex (Cx) and dorsal CA1 region of the hippocampus (HP) in mutants (B, G, arrowheads) relative to controls (A, E). No obvious GluN2B mRNA loss in ventral CA1, CA3, or dentate gyrus (DG) in a horizontal section of mutants (D) relative to controls (C). No differences in GluN2B mRNA in striatum (ST) (A, B), olfactory bulbs (C, D), or lateral (LA), basolateral (BLA), or central (Ce) amygdala nuclei (F, H). I, Loss of GluN2B protein levels in CA1 hippocampal tissue of mutants relative to controls (n = 4–12 per genotype). J, Loss of GluN2B and GluN1 but not GluN2A, CaMKIIα, or CaMKIIβ protein levels in cortical tissue of mutants relative to controls (n = 4–6 per genotype). Data are mean ± SEM normalized to levels in control genotype. *p < 0.05 versus control. Cb, Cerebellum; BS, brainstem; Con, control; Mut, mutant.

Figure 2.

Faster decaying NMDAR-mediated eEPSCs at CA1 hippocampus synapses. A, Decay time constant data showing decreased NMDAR-mediated EPSC tau for mutants compared with controls (n = 8–14 per genotype) (blue lines represent a single-exponential fit to the decay phase of the eEPSCs) (*p < 0.05 vs control). B, Peak amplitude of eEPSCs was not significantly different between genotypes (n = 8–14 per genotype). C, The selective GluN2B antagonist Ro 25-6981 (1 μm) reduced NMDAR-mediated eEPSCs in controls (Con), not mutants (Mut). The subunit-nonselective NMDAR antagonist dl-AP-5 (50 μm) reduced NMDAR-mediated eEPSCs equally in controls and mutants (n = 8–14 per genotype) (*p < 0.05 vs baseline/same genotype). Data are means ± SEM.

Figure 3.

Partially impaired hippocampal LTP and hippocampal LTD. A, HFS (2 100-Hz/s trains) produced subsaturating LTP in controls but not mutants (n = 5–6 per genotype) (arrow denotes stimulation). B, Representative traces and data showing three trains of HFS (2 100-Hz/s trains) produced saturating LTP in mutants and controls (n = 5–6 per genotype). C, Representative traces and data showing LFS (1 Hz/s) combined with the glutamate transporter blocker tPDC application produced LTD in controls, not mutants (n = 6–8 per genotype). D, LTD produced by LFS plus tPDC was blocked by application of AP-5 (n = 6–7 per genotype). Arrows denote LFS. Thin line denotes application of tPDC alone. Thick line denotes application of tPDC plus dl-AP-5. Time in C and D is from last stimulation. Data are means ± SEM.

Figure 4.

A, B, Decreased hippocampal neuron dendritic spine density. Example of labeled spines along apical dendrites ballistically labeled with fluorescent dye DiI in controls (A) and mutants (B). Scale bars, 7 μm. C, D, Mutants showed decreased spine density relative to controls as depicted at the level of individual dendrites (C) and neuron averages (D). E, F, Neither spine head width (E) nor spine length (F) distributions significantly differed between genotypes. n = 4–5 per genotype (10–18 neurons per genotype, 34–62 dendrites per genotype). Data are means ± SEM protrusions.

Figure 5.

Cognitive deficits. A, Mutants showed longer latencies to find a hidden platform in the Morris water maze than controls (n = 8 per genotype). B, Controls (Con) showed significant preference of the target quadrant over nontrained quadrants during a second, but not first, probe trial transfer test, whereas mutants (Mut) showed no preference on either (*p < 0.05 vs nontarget). C, Latencies to find a visible platform in the Morris water maze was not different between genotypes (n = 6 per genotype). D, Controls, but not mutants, showed significant spontaneous alternation (relative to chance) in a discrete-trial T-maze task (^#p < 0.05 vs 50%/chance) (n = 6 per genotype). E, Mutants showed significantly less conditioned (tone) freezing after trace fear conditioning (*p < 0.05 vs control) (n = 32–33 per genotype). F, Genotypes did not differ in conditioned (tone) freezing after delay fear conditioning (n = 9 per genotype). Data are means ± SEM.