Behavioral deficits and subregion-specific suppression of LTP in mice expressing a population of mutant NMDA receptors throughout the hippocampus

Abstract

The NMDA receptor (NMDAR) subunit GluN1 is an obligatory component of NMDARs without a known functional homolog and is expressed in almost every neuronal cell type. The NMDAR system is a coincidence detector with critical roles in spatial learning and synaptic plasticity. Its coincidence detection property is crucial for the induction of hippocampal long-term potentiation (LTP). We have generated a mutant mouse model expressing a hypomorph of the Grin1(N598R) allele, which leads to a minority (about 10%) of coincidence detection-impaired NMDARs. Surprisingly, these animals revealed specific functional changes in the dentate gyrus (DG) of the hippocampal formation. Early LTP was expressed normally in area CA1 in vivo, but was completely suppressed at perforant path-granule cell synapses in the DG. In addition, there was a pronounced reduction in the amplitude of the evoked population spike in the DG. These specific changes were accompanied by behavioral impairments in spatial recognition, spatial learning, reversal learning, and retention. Our data show that minor changes in GluN1-dependent NMDAR physiology can cause dramatic consequences in synaptic signaling in a subregion-specific fashion despite the nonredundant nature of the GluN1 gene and its global expression.

Figure 1.

GluN1^Rneo/+ mice express NMDAR GluN1^R subunits at low levels. (A) Genomic organization of the GluN1^Rneo allele with neo cassette in intron 18. Arrows indicate PCR primers for mRNA quantification plaque assay. Splicing that leads to the GluN1^R hypomorph is indicated for the PCR fragment. (B) Western blot from P0 brain membranes, probed with antibody against GluN1 C terminus. The weak GluN1^Rneo/Rneo signal is comparable to that obtained from GluN1^−/− preparations spiked with 5% GluN1^+/+ material (sample-loading control: ErbB-4). (C) Relative GluN1^R mRNA abundance in forebrain of adult GluN1^Rneo/+ mice (n = 5), determined by a plaque assay. (D) Western blot on adult brain homogenate. Antibodies against GluN1 N or C terminus revealed full-length GluN1 subunits and no truncated species in GluN1^Rneo/+. (E) Relative GluN1^R mRNA abundance of GluN1^R transcripts from individual CA1 pyramidal neurons or DG granule cells (n = 7 cells, each region), determined by plaque assay.

Figure 2.

Altered electrophysiological properties in the hippocampus of GluN1^Rneo/+ mice, in vivo. (A) Unaltered LTP in area CA1 after tetanic stimulation (n = 5, each genotype; ●, GluN1^Rneo/+; ○, GluN1^+/+). (B) Absence of LTP in the DG in GluN1^Rneo/+ animals (n = 9) after tetanic stimulation of the perforant path, in contrast to wild-type GluN1^+/+ (n = 8). (C) Stimulus-response curves for the population spike in the DG. Note strongly reduced population spike amplitude of GluN1^Rneo/+ animals compared with GluN1^+/+ wild-type mice (n = 7, each). Insets: Representative EPSP traces are included on the right (i) GluN1^+/+ and (ii) GluN1^Rneo/+; scales: 8 msec, 3 mV. (D) Stimulus-response curves for field EPSP in the DG are similar in GluN1^Rneo/+ and GluN1^+/+ (n = 7, each). (E) DG paired-pulse interaction. Note prolonged paired-pulse inhibition of population spike at short intervals and reduced facilitation at long intervals in GluN1^Rneo/+ animals compared with GluN1^+/+ wild-type mice, measured as percent change of the population spike height (n = 10, each). ISI corresponds to interstimulus interval. Representative traces are included on the right (i) GluN1^+/+ and (ii) GluN1^Rneo/+; scales: 10 msec, 4 mV. (F) Subthreshold stimulation resulted in identical paired-pulse facilitation of the EPSP in both genotypes, in the DG (n = 7, each). (G) Input/output curves of EPSP amplitude in the CA1 region from both genotypes (n = 5, each). (H) CA1 paired-pulse interaction. The inhibition and facilitation of the population spike was similar in both genotypes in area CA1 (n = 4, each).

Figure 3.

Impaired spatial learning of GluN1^Rneo/+ mice. (A,B) GluN1^Rneo/+ (n = 12) and wild-type GluN1^+/+ littermates (n = 13) were trained to a fixed hidden platform position in a watermaze for seven consecutive days (acquisition, four trials a day), followed by 6 d, to a platform position in the opposite pool quadrant (reversal, after 21-d interval). (B) Identical escape latencies for both genotypes at the beginning of the experiment, as shown by the results from individual trials on training day 1. (C) (○) Wild-type GluN1^+/+, (n = 13) and (●) GluN1^Rneo/+ mice, (n = 12) were trained for 6 d with four trials per day to locate the platform marked by a local visual cue, prior to the training for the hidden platform. The two groups performed equivalently well at day 1 (ANOVA, P > 0.65), and by training day 6 (ANOVA, P > 0.33). The differences between the groups during days 2–5 (ANOVA, P = 0.0007, 0.005, 0.01, and 0.0002, respectively) suggest that the mutants were slower learners than their wild-type littermates during this period, but this difference was insignificant by the end of the task. (D,E) Probe tests (PT1 and PT2) were performed (without platform) after training days 7 and 13, respectively. GluN1^+/+ mice spent more time in the TQ than GluN1^Rneo/+ mice. (F) Representative swim paths from PT1. (TQ) Training quadrant; (AL) adjacent left; (OQ) opposite; (AR) adjacent right; (+) start of swim path; (●) training position of platform.

Figure 4.

Normal performance of GluN1^Rneo/+ mice in motor-coordination and novel object recognition tasks. (A) Rotarod test. Latencies of GluN1^Rneo/+ (n = 12) and wild-type GluN1^+/+ littermates (n = 8) over three training days. No difference in performance was observed between the genotypes over the 3 d of training. (B) Novel object recognition. The two genotypes showed equivalent performance when tested shortly (1-min interval) after training. Neither genotype retained a preference for the novel object when tested 1 h after training. GluN1^Rneo/+ (n = 8) and wild-type GluN1^+/+ littermates (n = 6).

Figure 5.

Impaired place recognition of GluN1^Rneo/+ mice. (A) Diagram showing location of the “annulus” swim corridor (16-cm wide, inner ring) within the watermaze. The inaccessible portions of the watermaze (outer ring and inner circle) are shown in gray. It is notionally divided into six segments, one of which is designated the goal segment. The on-demand Atlantis platform comes up after one complete swimming lap of the corridor. (B) Mean swim paths (m), escape latencies (s), and swim speed (cm/sec) for the 5 d of training in the place recognition task. While the swim paths were necessarily the same, mutants took longer to escape and swam more slowly. (C) Percent time spent in the goal segment of the annulus during PT1 (1 d after training) and PT2 (5 d after completion of training). The groups differ on PT1 and only the wild-type mice were above chance. (D) Removal of the inner corridor created a smaller open field watermaze for cue task training (open inner circle). (E) Escape latencies over the four trials of training showed no difference between groups.