Reduced expression of the NMDA receptor-interacting protein SynGAP causes behavioral abnormalities that model symptoms of Schizophrenia

Abstract

Abnormal function of NMDA receptors is believed to be a contributing factor to the pathophysiology of schizophrenia. NMDAR subunits and postsynaptic-interacting proteins of these channels are abnormally expressed in some patients with this illness. In mice, reduced NMDAR expression leads to behaviors analogous to symptoms of schizophrenia, but reports of animals with mutations in core postsynaptic density proteins having similar a phenotype have yet to be reported. Here we show that reduced expression of the neuronal RasGAP and NMDAR-associated protein, SynGAP, results in abnormal behaviors strikingly similar to that reported in mice with reduced NMDAR function. SynGAP mutant mice exhibited nonhabituating and persistent hyperactivity that was ameliorated by the antipsychotic clozapine. An NMDAR antagonist, MK-801, induced hyperactivity in normal mice but SynGAP mutants were less responsive, suggesting that NMDAR hypofunction contributes to this behavioral abnormality. SynGAP mutants exhibited enhanced startle reactivity and impaired sensory-motor gating. These mice also displayed a complete lack of social memory and a propensity toward social isolation. Finally, SynGAP mutants had deficits in cued fear conditioning and working memory, indicating abnormal function of circuits that control emotion and choice. Our results demonstrate that SynGAP mutant mice have gross neurological deficits similar to other mouse models of schizophrenia. Because SynGAP interacts with NMDARs, and the signaling activity of this protein is regulated by these channels, our data in dicate that SynGAP lies downstream of NMDARs and is a required intermediate for normal neural circuit function and behavior. Taken together, these data support the idea that schizophrenia may arise from abnormal signaling pathways that are mediated by NMDA receptors.

Figure 1. SynGAP heterozygous mutants display elevated stereotypy

A. Three month old SynGAP wt (n=14) and het (n=14) mice were placed in the open field environment for two hours. Stereotypic counts were broken down by five-minute blocks. SynGAP hets demonstrated elevated movements during each time-bin. B. Total stereotypic counts for each genotype over the entire two hour test session; One-way ANOVA. C. A different cohort of WT (n=10) and Het (n=10) mice were exposed to the open field environment for thirty minutes for five consecutive days. Stereotypy was broken down by five-minute intervals. D. This same cohort of mice was IP injected with 10ml/kg of saline thirty minutes prior to placement into the open field. Stereotypy was measured over a sixty minute period. E. These same mice were injected with 0.2mg/kg MK-801 thirty minutes prior to placement in the open field. F. Comparison of total stereotypic counts from both genotypes between saline and Mk-801 injections (n=10 per group). A 2 × 2 ANOVA determined an interaction between each genotype and drug treatment group. To determine significance between individual groups, we performed a post-hoc paired samples test between saline and MK-801 within genotypes, and an independent samples test between genotypes within the same drug treatment. *p < 0.05, **p<0.01

Figure 2. SynGAP Hets display enhanced horizontal locomotion

A. Ambulatory distance of WT (N=11) and Heterozygous (N=12) SynGAP mutants at 15 minute intervals over a two-hour period. ANOVA. B. WT (n=10) and Het (n=9) mice were exposed to the open field environment for thirty minutes for five consecutive days. Ambulatory distance was summed over a thirty minute trial period. RM ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001 C. Representative ambulatory vector for either a WT or a Het mouse over a thirty minute period. D. Thigmotaxis calculation for each genotype (WT = 10; Het = 11). This calculation was performed for the mice in the open field shown in panel A. To determine thigmotaxis, a zonal analysis was performed over the first fifteen minutes of the open field fun. The total distance traveled in the periphery of the chamber relative to the distance traveled in the center was calculated for each minute. Each value was averaged over a five minute period and plotted as three blocks. We determined that an interaction between the genotypes was present by performing a repeated measures ANOVA and significance was indicated specifically at the second time point (F_{(1, 20)} = 10.83, p < 0.01). E. SynGAP WT (n=9) and heterozygous (n=7) mice between three and five weeks of age were placed in the open field and ambulatory distanced were measured. ANOVA; *p < 0.05 F. 3–4 month old WT (n=10) and Het (n=10) mice were IP injected with saline thirty minutes prior to placement into the open field. Ambulatory distance was measured over a sixty minute period. One week later, these same mice were injected with 0.2mg/kg MK-801 thirty minutes prior to placement in the open field. G. Comparison of total ambulatory distance from both genotypes between saline and MK-801 injections (n=10 per group). To determine significance between genotype and drug treatment, we used a 2 × 2 ANOVA. To determine significance between individual groups, we performed a paired samples t-test between saline and MK-801 within genotypes, and an independent samples t-test between genotypes within the same drug treatment. *p < 0.05, **p < 0.01 H. WT (n=9) and Het (n=9) mice were injected with varying doses of vehicle (v) or clozapine (d; 0.3, 1.0, 2.0 mg/Kg) thirty minutes before placement into the open field. Total horizontal distance over this period was determined for each animal. To determine significance between genotype and each drug treatment, we used a 2 × 2 ANOVA [(0.3 mg/kg - genotype: F_{(1, 17)} = 8.92, p < 0.05; Drug: F_(1,17) = 0.24, p = 0.64) (1.0 mg/kg - genotype: F_{(1, 17)} = 7.30, p < 0.05; Drug: F_(1,17) = 7.57, p < 0.05) (2.0 mg/kg - genotype: F_{(1, 17)} = 4.66, p = 0.07; Drug: F_(1,13) = 36.0, p < 0.001). If an interaction was observed, we performed a post hoc independent samples test to determine significance between genotypes within the same drug treatment group, and a paired samples test to determine significance within a genotype between saline and drug treatments.

Figure 3. Reduced SynGAP expression results in enhanced startle reactivity and abnormal sensory-motor gating

A. SynGAP WT (n=11) and Heterozygous (n=11) mutants were tested for startle responses to a 120 dB white noise burst. These mice were naïve to the startle stimulus. ANOVA, * p < 0.05 B. SynGAP WT (n=14) and Heterozygous (n=11) mutants were tested for startle responses to a series of startle responses of increasing amplitude. The startle stimuli were presented at random and each was presented at least ten times. C. WT (n=8) and Het (n=6) mice were tested for prepulse inhibition. Mixed factor ANOVA, **p < 0.01; D. WT (black/grey bars, n=8) and Het (white/grey bars, n=5) mice were injected with vehicle or 2 mg/Kg clozapine (grey bars) thirty minutes before testing in the startle chamber. There was no significant effect of clozapine on PPI in either genotype.

Figure 4. SynGAP heterozygous mutants lack short-term social memory and exhibit social isolation

A. Both genotypes of SynGAP mice (N=7 per group) were exposed to the social interaction apparatus for ten minutes that contained the wire cages. There were no stranger mice present during this habituation session. Both genotypes preferred to spend time in the each end of the apparatus (L, R). There was no significant left-right place preference in either genotype. Wilcoxon Signed Ranks Test B. WT (n=7) or Het (n=7) mice were allowed to explore the social interaction apparatus for ten minutes (600 seconds). A stranger conspecific was placed inside one of the wire cages (mouse) while the other end of the apparatus contained an empty, but identical, wire cage (object). The time spent in each end of the apparatus is displayed for each genotype. Both genotypes displayed a clear preference for the conspecific. Wilcoxon Signed Ranks Test C. WT (N=7) or Het (n=7) mice were allowed to explore the social interaction apparatus for ten minutes. The test mouse was first habituated to a conspecific over ten minutes (same). An unfamiliar conspecific was then added (different) and the time spend in each section of the apparatus was measured (600 seconds total). WT mice displayed a clear preference for the novel conspecific while SynGAP Hets did not. Wilcoxon Signed Ranks Test D. Both genotypes (n=6 per group) were tested for their ability to find buried food in a test cage. Mice were first food-restricted to 85% of normal body weight and then presented with a food reward that was clearly visible. The next day, these food rewards were buried in the bedding of a novel test cage and the latency to find the reward was measured. ANOVA E. WT and Het mice were placed into the social interaction apparatus and allowed to explore the entire environment for three consecutive ten-minute sessions. The wire cages were present on each end of the apparatus during the entire test. The time spent in the center compartment was measured for each genotype (n=7/group). F. The time in the center compartment was measured during each ten-minute session of the social recognition test (Block 1 = Habituation; Block 2 = Social Interaction; Block 3 = Social Recognition). Differences between genotypes at each block were demined by a Mixed Factor ANOVA (n=7/group). *p < 0.05 G. Total number of chamber entries was recorded during the third block of the sociability test (n=7/group). A chamber entry was defined as a complete crossing form one compartment into the other. ANOVA.

Figure 5. SynGAP mutants have abnormal behavioral responses to emotional cues

A. SynGAP WT and Het mice were trained for contextual plus cued fear conditioning. Twenty-four hours after training, mice (n=21 WT/n= 19 Het) were replaced into the testing chamber and freezing was scored over each minute of the test. ANOVA B. The same mice were also tested for freezing in response to an auditory cue. Left, 28 hours after training, mice were placed into a novel context and allowed to explore for three minutes. Freezing was measured over this entire period. Right, After the three minute acclimation period, the conditioning tone was presented for three minutes and freezing in response to the CS presentation was scored (n=17 WT/n= 10 Het). ANOVA, **p<0.001 C. Left, Activity of WT (n=17) and Het (n=10) mice was recorded in the thirty seconds prior to (pre-tone) and immediately after (tone) the first conditioning tone. We determined an interaction only within genotypes by a 2×2 ANOVA (genotype: F(1,26) = 28.5, p < 0.001; activity: F(1,26) = 3.01, p = 0.121). D. WT (n=8) and Het (n=6) mice were put in the elevated T-maze for fifteen minutes. A computerized tracking device monitored both the time spent in each area of the maze and the number of entries in each location (i.e. open arm, closed arm, and hub). Differences in arm time and arm entries were determined by an ANOVA with repeated measures.

Figure 6. SynGAP mutants have working memory deficits

A. Data describes the performance of each genotype during the habituation (number of days until animal took six pellets in 10 minutes) and training portion of the task (see methods). B. SynGAP WT (n=8) and Het (n=7) mice were tested Non-Match to Place performance at five and thirty-second delays. The grey bar represents the performance of each group during the training sessions (3.4 second delay). * ANOVA with repeated measures, p < 0.005; ^# one-sample T-test, p < 0.005. One sample T-Test values were 75% (0.75) for training criterion and 50% (0.50) for random chance. C. Left, Average choice latency indicates time taken to enter the arm during non-matching. This value includes the 2.4 seconds it takes for the door to fully open in our maze. Right, Average test session duration for each animal (average over three days)