Lack of neuronal nitric oxide synthase results in attention deficit hyperactivity disorder-like behaviors in mice

Abstract

Nitric oxide (NO) is an important molecule for the proper development and function of the central nervous system. In this study, we investigated the behavioral alterations in the neuronal NO synthase knockout mice (NOS1 KO) with a deficient NO production mechanism in the brain, characterizing it as a potential rodent model for attention deficit hyperactivity disorder (ADHD). NOS1 KO exhibited higher locomotor activity than their wildtype counterparts in a novel environment, as measured by open field (OF) test. In a 2-way active avoidance paradigm (TWAA), we found sex-dependent effects, where male KO displayed deficits in avoidance and escape behavior, sustained higher incidences of shuttle crossings, and higher incidences of intertrial interval crossings, suggesting learning, and/or performance impairments. On the other hand, female KO demonstrated few deficits in TWAA. Molsidomine (MSD), a NO donor, rescued TWAA deficits in male KO when acutely administered before training. In a passive avoidance paradigm, KO of both sexes displayed significantly shorter step-through latencies after training. Further, abnormal spontaneous motor activity rhythms were found in the KO during the dark phase of the day, indicating dysregulation of rhythmic activities. These data indicate that NOS1 KO mimics certain ADHD-like behaviors and could potentially serve as a novel rodent model for ADHD.

Figure 1

OF test indicated that NOS1 KO were hyperactive in a novel environment, but not significantly more anxious. KO showed significantly increased locomotor activity as measured by ambulatory time, distance travel, and ambulatory activity counts (a–c). No significant difference was found in the center time, stereotypic or vertical activities (d–f). Data represent means ± SEM. Significance level: *p < .05, one-way ANOVA followed by post hoc Bonferroni test. Sample size: n = 11 for WT (5 male and 6 female); n = 14 for HT (6 male and 8 female); n = 21 for KO (9 male and 12 female).

Figure 2

NOS1 KO, notably males, showed abnormal performances when compared to WT in TWAA paradigm, namely the percentage of successful TWAA Response to tone (a, e), the percentage of failure to escape the foot shock (Escape Failure, b, f), number of EXP Crossing (c, g), and number of ITI Crossing (d, h). Males and females are plotted separately. MSD (20mg/kg, daily) treatment rescued the deficits of TWAA Response and Escape Failure in male KO, while resulting in paradoxical impairing effects in male WT. Significance level: *p < .05, significant difference between WT-Sal and KO-Sal was detected by one-way ANOVA followed by post hoc Dunnett’s t-tests; ^#p < .05, significant difference between WT-Sal and KO-MSD was detected by one-way ANOVA followed by post hoc Dunnett’s t-tests; ^×p < .05, significant difference between WT-Sal and WT-KO was detected by one-way ANOVA followed by post hoc Dunnett’s t-tests. Sample size: n = 18 for WT receiving saline treatment (9 male and 9 female); n = 14 for WT receiving MSD treatment (7 male and 7 female); n = 16 for KO receiving saline treatment (8 male and 8 female); n = 14 for KO receiving MSD treatment (7 male and 7 female).

Figure 3

NOS1 KO showed deficits in PA retention test. NOS1 KO exhibited shorter step-through latency in retention tests as compared to their WT counterparts. Data represent means ± SEM. Significance level: *: p < .05; **: p < .01; ***: p < .001, paired or unpaired t-test. Sample size: n = 24 for WT (12 male and 12 female); n = 19 for KO (9 male and 10 female).

Figure 4

NOS1 KO showed abnormal SMAR in the dark phase of the day. (a) The peak frequency in the slow-wave band (left) and the % active time (right) were analyzed. (b–c) Representative results of a WT and a KO mouse, showing the output of FFT analysis (b) and the raw activity indexes (c). Data represent means ± SEM. Significance level **: p < .01, Mann-Whitney U test. Sample size: n = 14 for WT (7 male and 7 female); n = 14 for KO (7 male and 7 female).

Figure 5

Summary of evidence supporting the hypothesis that dysfunction of NOS1 and NO system may result in ADHD-like behaviors.