Severe impairment of NMDA receptor function in mice carrying targeted point mutations in the glycine binding site results in drug-resistant nonhabituating hyperactivity

Abstract

NMDA receptor hypofunction has been implicated in the pathophysiology of schizophrenia, and pharmacological and genetic approaches have been used to model such dysfunction. We previously have described two mouse lines carrying point mutations in the NMDA receptor glycine binding site, Grin1(D481N) and Grin1(K483Q), which exhibit 5- and 86-fold reductions in receptor glycine affinity, respectively. Grin1(D481N) animals exhibit a relatively mild phenotype compatible with a moderate reduction in NMDA receptor function, whereas Grin1(K483Q) animals die shortly after birth. In this study we have characterized compound heterozygote Grin1(D481N/K483Q) mice, which are viable and exhibited biphasic NMDA receptor glycine affinities compatible with the presence of each of the two mutated alleles. Grin1(D481N/K483Q) mice exhibited a marked NMDA receptor hypofunction revealed by deficits in hippocampal long-term potentiation, which were rescued by the glycine site agonist d-serine, which also facilitated NMDA synaptic currents in mutant, but not in wild-type, mice. Analysis of striatal monoamine levels revealed an apparent dopaminergic and serotonergic hyperfunction. Behaviorally, Grin1(D481N/K483Q) mice were insensitive to acute dizocilpine pretreatment and exhibited increased startle response but normal prepulse inhibition. Most strikingly, mutant mice exhibited a sustained, nonhabituating hyperactivity and increased stereotyped behavior that were resistant to suppression by antipsychotics and the benzodiazepine site agonist Zolpidem. They also displayed a disruption of nest building behavior and were unable to perform a cued learning paradigm in the Morris water maze. We speculate that the severity of NMDA receptor hypofunction in these mice may account for their profound behavioral phenotype and insensitivity to antipsychotics.

Fig. 1.

Glycine and glutamate concentration–response curves from Grin1^D481N/K483Q mouse hippocampal neurons. A, Mean glycine concentration–response data fromGrin1^D481N/K483Q mice. Mean ± SE peak currents elicited by the application of 100 μmNMDA in the presence of increasing concentrations of glycine (n = 7) are expressed as a function of the maximum peak response derived from a fitted curve of the peak glycine concentration–response data for each individual neuron, using the 2× two-equivalent binding site model. A curve fit with the 2× two-equivalent binding site model yieldedmK_D values and relative amplitudes of 0.22 μm (16%) and 3.41 μm (84%) for the high- and low-affinity components, respectively. The dashed line shows a representative monophasic glycine concentration–response curve from control wild-type hippocampal neurons (mK_D = 38 nm) (Kew et al., 2000). B, Mean glutamate concentration–response data fromGrin1^D481N/K483Q mice. A curve fit with the two-equivalent binding site model yielded anmK_D value of 3.2 μm. Mean ± SE peak currents elicited by the application of increasing concentrations of glutamate in the continual presence of 100 μm glycine and 10 μm NBQX (n = 3) are expressed as a function of the maximum peak response derived from a fitted curve of the peak glutamate concentration–response data for each individual neuron with the two-equivalent binding site model. The dashed line shows representative glutamate concentration–response data from control wild-type hippocampal neurons (mK_D = 1.9 μm) (Kew et al., 2000).

Fig. 2.

Reduced NMDA receptor glycine site occupancy in brain slices from Grin1^D481N/K483Qmice. A, NMDA-induced population depolarizations in cortical wedges from wild-type (open bars,n = 22) andGrin1^D481N/K483Q mice (solid bars, n = 7–14). Mean ± SE depolarizations produced by the application of 20 μmNMDA in the presence of increasing concentrations ofd-serine are expressed as a percentage increase relative to the depolarization produced by the application of 20 μm NMDA alone in each individual slice (i.e., 20 μm NMDA alone, 0%). The relative increase in response amplitude after the addition of d-serine was significantly greater inGrin1^D481N/K483Q, but not wild-type, mice (*p < 0.05; ***p < 0.001; two-tailed t test). B, Representative traces illustrating NMDA receptor-mediated EPSCs recorded from CA1 pyramidal neurons in hippocampal slices fromGrin1^D481N/K483Q and wild-type mice in the absence (black lines) and presence (gray lines) of 100 μm d-serine. EPSCs recorded fromGrin1^D481N/K483Q, but not wild-type, mice were potentiated significantly in the presence ofd-serine. C, Theta burst-induced LTP in hippocampal slices from wild-type andGrin1^D481N/K483Q mice in the absence (open circles, n = 8; open squares, n = 8) and presence (filled circles, n = 8;filled squares, n = 7) of 100 μm d-serine. Where added, d-serine was present from 30 min before theta burst stimulation to the end of the experiment. Mean ± SE field EPSP slopes are expressed as a percentage of baseline values recorded 10 min before theta burst stimulation.

Fig. 3.

A, The mean weight (gm) ± SE of wild-type (open circles, n = 20) and Grin1^D481N/K483Q(filled circles, n = 13) mice over weekly testing periods. B, Spontaneous locomotor activity (mean mobile counts ± SE) in novel activity cages of wild-type (open circles, n = 14) andGrin1^D481N/K483Q (filled circles, n = 14) mice. The data are presented in 5 min time bins over a 1 hr period; theinset represents the total activity in 1 hr (unpairedt test; **p < 0.01 vs wild-type).C, Photographs of representative nests built by a wild-type mouse (left) and aGrin1^D481N/K483Q mouse (right). The bar graph represents the percentage of mice with completed nests 24 hr after the placement of a folded tissue in the home cage. D, Distance traveled (in centimeters) and stereotypy counts of wild-type (open circles,n = 12) andGrin1^D481N/K483Q (filled circles, n = 12) mice expressed as means ± SE per hour over a 24 hr period. The white bar below the x-axis represents the light phase (6:00 A.M. to 6:00 P.M.), and the black bar represents the dark phase (6:00 P.M. to 6:00 A.M.) of testing.

Fig. 4.

Effect of dizocilpine (0.1, 0.3 mg/kg) in wild-type (n = 12) andGrin1^D481N/K483Q(n = 7) mice on the total distance (in centimeters) traveled (A) and stereotypy counts (B) in a 90 min period. Effect of amphetamine (0.3, 1, 3 mg/kg) in wild-type (n = 11) andGrin1^D481N/K483Q(n = 5) mice on the total distance (in centimeters) traveled (C) and stereotypy counts (D) in a 1 hr period. Data are expressed as means ± SE; repeated measures design. *p < 0.05, **p < 0.01 dose versus respective vehicle group; ^#p < 0.05,^##p < 0.01Grin1^D481N/K483Q versus wild type (vehicle-treated). WT, Wild-type mice;TG, Grin1^D481N/K483Qmice.

Fig. 5.

Effect of clozapine (0.1, 0.3, 3 mg/kg) in wild-type (n = 13) andGrin1^D481N/K483Q(n = 12) mice on the total distance (in centimeters) traveled (A) and stereotypy counts (B) in a 1 hr period. Effect of haloperidol (0.03, 0.1, 0.3 mg/kg) in wild-type (n = 10) andGrin1^D481N/K483Q(n = 6) mice on the total distance (in centimeters) traveled (C) and stereotypy counts (D) in a 1 hr period. Effect of M100907 (0.003, 0.03, 0.3 mg/kg) in wild-type (n = 12) andGrin1^D481N/K483Q(n = 11) mice on the total distance (in centimeters) traveled (E) and stereotypy counts (F) in a 1 hr period. Effect of Zolpidem (3, 10 mg/kg) in wild-type (n = 11) andGrin1^D481N/K483Q(n = 5) mice on the total distance (in centimeters) traveled (G) and stereotypy counts (H) in a 1 hr period. Data are expressed as means ± SE; repeated measures design. *p< 0.05, **p < 0.01 dose versus respective vehicle group; #p < 0.05, ##p < 0.01Grin1^D481N/K483Q versus wild type (vehicle-treated). WT, Wild-type mice;TG, Grin1^D481N/K483Qmice.

Fig. 6.

A, Startle amplitude (means ± SE) of wild-type (white bar) andGrin1^D481N/K483Q (black bar) after no stimulus (NS); shown are pulses of 74, 82, and 90 dB (P74, P82, P90) and stimulus threshold of 110 dB (ST110). B, Percentage of prepulse inhibition at prepulses of 74, 82, and 90 dB, followed by a 110 dB pulse. C, Percentage of prepulse inhibition at prepulse (90 dB), followed by a pulse (110 dB) after different interstimulus intervals of 30, 100, and 300 msec. Data are expressed as means ± SE; *p < 0.05 versus respective wild-type group.

Fig. 7.

A, Path length (in centimeters) traveled to find a visible cued platform in the water maze (means of 3 trials ± SE) per session (two sessions per day) by wild-type (open circles, n = 10) andGrin1^D481N/K483Q mice (filled circles, n = 8).B, Swim paths of a representative wild-type mouse (one trial from each session). C, Swim paths of representative Grin1^D481N/K483Q mouse (one trial from each session).