Comprehensive behavioral analysis of heterozygous Syngap1 knockout mice

Abstract

Aims: Synaptic Ras GTPase-activating protein 1 (SYNGAP1) regulates synaptic plasticity through AMPA receptor trafficking. SYNGAP1 mutations have been found in human patients with intellectual disability (ID) and autism spectrum disorder (ASD). Almost every individual with SYNGAP1-related ID develops epilepsy, and approximately 50% have ASD. SYNGAP1-related ID is estimated to account for at least 1% of ID cases. In mouse models with Syngap1 mutations, strong cognitive and affective dysfunctions have been reported, yet some findings are inconsistent across studies. To further understand the behavioral significance of the SYNGAP1 gene, we assessed various domains of behavior in Syngap1 heterozygous mutant mice using a behavioral test battery.

Methods: Male mice with a heterozygous mutation in the Syngap1 gene (Syngap1^-/+ mice) created by Seth Grant's group were subjected to a battery of comprehensive behavioral tests, which examined general health, and neurological screens, rotarod, hot plate, open field, light/dark transition, elevated plus maze, social interaction, prepulse inhibition, Porsolt forced swim, tail suspension, gait analysis, T-maze, Y-maze, Barnes maze, contextual and cued fear conditioning, and home cage locomotor activity. To control for type I errors due to multiple-hypothesis testing, P-values below the false discovery rate calculated by the Benjamini-Hochberg method were considered as study-wide statistically significant.

Results: Syngap1^-/+ mice showed increased locomotor activity, decreased prepulse inhibition, and impaired working and reference spatial memory, consistent with preceding studies. Impairment of context fear memory and increased startle reflex in Syngap1 mutant mice could not be reproduced. Significant decreases in sensitivity to painful stimuli and impaired motor function were observed in Syngap1^-/+ mice. Decreased anxiety-like behavior and depression-like behavior were noted, although increased locomotor activity is a potential confounding factor of these phenotypes. Increased home cage locomotor activity indicated hyperlocomotor activity not only in specific behavioral test conditions but also in familiar environments.

Conclusion: In Syngap1^-/+ mice, we could reproduce most of the previously reported cognitive and emotional deficits. The decreased sensitivity to painful stimuli and impaired motor function that we found in Syngap1^-/+ mice are consistent with the common characteristics of patients with SYNGAP-related ID. We further confirmed that the Syngap1 heterozygote mouse recapitulates the symptoms of ID and ASD patients.

Keywords: SYNGAP1; autism spectrum disorder; intellectual disability; motor function; nociception.

Figure 1

General health and neurological screen, motor learning, and pain sensitivity between genotypes. A, Body weight; B, body temperature; C, grip strength; D, latency to fall in wire hang test; E, latency to fall in the rotarod test; and F, latency of the first fore‐ or hind paw response in the hot plate test. Data represent the mean ± SEM. The P‐values indicate genotype effects in a one‐way ANOVA (A‐D, and F), or two‐way repeated measures ANOVA (E)

Figure 2

Increased locomotor activity of Syngap1 ^−/+ mice in open field test. A, Total distance traveled; B, vertical activity; C, time spent in the center area; D, stereotypic behavior counts are represented. Data represent the mean ± SEM. The P‐values indicate genotype effects in two‐way repeated measures ANOVA

Figure 3

Anxiety‐related behaviors of Syngap1 ^−/+ mice observed in elevated plus maze and light/dark transition test. (A‐D) Light/dark transition test: A, distance traveled in the light/dark compartments; B, number of light/dark transitions; C, latency to enter the light compartment; and D, time spent in the light compartment. (E‐H) Elevated plus maze test: E, distance traveled; F, percentage of entries into open arms; G, percentage of time spent in open arms; H, number of arm entries; I, percentage of mice dropped from the maze. Data represent the mean ± SEM. The P‐values in panels (A‐I) indicate genotype effects in one‐way ANOVA. The P‐value of (I) was evaluated using a chi‐square test. Only the data of the mice that completed the session without falling (controls, n = 14; mutants, n = 12) were included in the statistical analysis in E‐H

Figure 4

Sociability and social novelty preference of Syngap1 ^−/+ mice. (A‐D) Social interaction test in novel environments: A, total duration of contacts; B, number of contacts; C, mean duration per contact; and D, total distance traveled. (E‐J) Crawley's sociability and social novelty preference test: E, time spent around the cage, and F, total distance traveled in the sociability test; G, time spent around the cage, and H, total distance traveled in the social novelty preference test. Data represent the mean ± SEM. The P‐values in (A‐D, F, and H) indicate genotype effects in one‐way ANOVA. The P‐values in panels (E) and (G) represent the genotype effects (controls vs mutants) or side effects (empty side vs stranger 1 side, or stranger 1 side vs stranger 2 side) in one‐way ANOVA

Figure 5

Decreased prepulse inhibition of Syngap1 ^−/+ mice. A, Startle amplitude and B, percent of prepulse inhibition were tested. Data represent the mean ± SEM. The P‐values indicate genotype effects in two‐way repeated measures ANOVA that was separately performed in experiment with different startle sound level

Figure 6

Decreased immobility of Syngap1 ^−/+ mice in the tests for depression‐like behavior. A, Percentage of immobility time on day 1 and day 2 in a Porsolt forced swimming test. B, Percentage of immobility time in the tail suspension test. Data represent the mean ± SEM. The P‐values indicate genotype effects in two‐way repeated measures ANOVA

Figure 7

Impaired working memory and increased locomotor activity of Syngap1 ^−/+ mice observed in Y‐maze and T‐maze. (A‐C) T‐maze spontaneous alternation test: A, percentage of correct responses; B, latency to complete a session; C, distance traveled to complete a session. (D‐G) Y‐maze test: D, number of entries; E, total alterations; F, number of alterations as a percentage of total entries; and G, total distance traveled. Data represent the mean ± SEM. The P‐values indicate genotype effects in two‐way repeated measures ANOVA (A‐C), or one‐way ANOVA (D‐G)

Figure 8

Impaired spatial reference memory of Syngap1 ^−/+ mice in the Barnes maze. A, Number of errors before reaching the target hole; B, latency to reach the target hole; C, distance to reach the target hole; and D, number of omission errors before reaching the target hole are shown. E, Time spent around each hole in the probe trial conducted 1 d (left) and 1 mo (right) after the last training session. The P‐values indicate genotype effects in two‐way repeated measures ANOVA (A‐D), or one‐way ANOVA (E)

Figure 9

Contextual and cued fear memory in Syngap1 ^−/+ mice. (A) Shock sensitivity measured by the distance traveled during the shock. Percentage of freezing time during: (B) conditioning, (C, top) context testing, (D, top) cued testing with altered context, (E, top) context testing after 30 d, and (F, top) cued testing with altered context after 30 d. Each data point in the figure panels (B) and (C‐F, top) indicates percentage of freezing in each 1‐min bin. Distance traveled in: (C, bottom) context testing, (D, bottom) cued testing with altered context, (E, bottom), context testing after 30 d, and (F, bottom) cued testing with altered context after 30 d. Each data point in figure panels (C‐F, bottom) indicates the distance traveled in each 1‐min bin. Data represent the mean ± SEM. The P‐values indicate genotype effects in two‐way repeated measures ANOVA. The horizontal black bars indicate the time during which the tone stimuli were administered

Figure 10

Elevated locomotor activity and decreased social activity in Syngap1 ^−/+ mice in home cage. (A‐B) Social interaction in home cage as indicated by (A) mean number of particles, and (B) activity levels. (C) Home cage locomotor activity of single mouse. Data represent the mean ± SEM. The P‐values indicate genotype effects in two‐way repeated measures ANOVA. The three P‐values in each panel (A‐C) represent the genotype effects (controls vs mutants) in two‐way repeated measures ANOVA for the activity levels of whole day, day, or night, from top row