Behavioral Neuroscience 2015, Vol. 129, No. 1, 50 – 61

© 2015 American Psychological Association 0735-7044/15/$12.00 http://dx.doi.org/10.1037/bne0000031

Lack of Neuronal Nitric Oxide Synthase Results in Attention Deficit Hyperactivity Disorder-Like Behaviors in Mice Yudong Gao and Scott A. Heldt

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University of Tennessee Health Science Center Neuroscience Institute 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. Keywords: nitric oxide, NOS1, ADHD, learning, hyperactivity Supplemental materials: http://dx.doi.org/10.1037/bne0000031.supp

(ADHD) in humans (Lesch, Merker, Reif, & Novak, 2013). ADHD is a psychiatric disorder with hyperactivity, inattention, and impulsivity as core symptoms (American Psychiatric Association, 2000), and is often comorbid with learning disability (Semrud-Clikeman & Bledsoe, 2011), substance abuse (Wilens, Biederman, Mick, Faraone, & Spencer, 1997), epilepsy (Cohen et al., 2013), and other psychiatric conditions such as anxiety disorders (Cak et al., 2013) and disturbance of circadian rhythm (Kooij & Bijlenga, 2013). Neuronal NO synthase (NOS1) is a key enzyme responsible for the neuronal production of intracellular signaling molecule NO. Past study shows that 28% of adult ADHD patients were found to be homozygous for a risk allele in the NOS1 promoter region (termed ex1f-VNTR) that reduces NOS1 expression. This allele is prominently linked with altered prefrontal cortex (Reif et al., 2009) and ventral striatal (Hoogman et al., 2011) functions, which are both implicated in impulsive and aggressive behaviors associated with ADHD. Several animal models have been used to investigate the underlying neurobiological deficits of ADHD, including spontaneously hypertensive rat model (Meneses et al., 2011; Sagvolden, 2000), dopamine transporter knockout mouse (Gainetdinov, Caron, & Lombroso, 2001; Gainetdinov et al., 1999; Yamashita et al., 2013), and perinatal hypothyroidism rat (Negishi et al., 2005). These models use behavioral paradigms including open field test (OF), passive/two-way active avoidance (PA/TWAA), cliff avoidance test, five choice serial reaction time (RT) test, and spontaneous motor activity rhythm test (SMAR) to examine hyperactivity, learning impairment, inattention, impulsivity, and deficits in spontaneous activities as indicators of ADHD-related behavioral phenotypes in rodents. A number of past findings also suggest that NOS1 knockout mice (NOS1 KO) could be a candidate model for ADHD (Franke,

Nitric oxide (NO) modulates a variety of physiological processes, such as neurotransmission, development, plasticity, and neuronal cell death (Li et al., 2013; Prast & Philippu, 2001; Tricoire & Vitalis, 2012). Molecular lines of evidence suggest that the NO system interacts extensively with glutaminergic and monoaminergic signaling pathways (Christopherson, Hillier, Lim, & Bredt, 1999; Duchemin, Neff, & Hadjiconstantinou, 2010; Kiss & Vizi, 2001; Kiss, Zsilla, & Vizi, 2004; Zabel et al., 2002). Experiments that pharmacologically manipulate NO signaling pathways also demonstrate the functional role of NO in learning and memory (Chien, Liang, & Fu, 2008; Komsuoglu-Celikyurt et al., 2011; Küçükatay, Haciog˘lu, Ozkaya, Ag˘ar, & Yargiçog˘lu, 2009; Pitsikas et al., 2001). Recent findings suggest alterations in NO signaling pathways may be associated with attention deficit hyperactivity disorder

Yudong Gao and Scott A. Heldt, Department of Anatomy and Neurobiology, University of Tennessee Health Science Center Neuroscience Institute. Financial support was provided by National Institutes of Health (MH086727), Brain & Behavior Research Foundation (formerly National Alliance for Research in Schizophrenia and Affective Disorders), and a University of Tennessee Health Science Center base grant. The present work also benefited from Francesca-Fang Liao, who provided the neuronal nitric oxide synthase knockout strain; Brittany Wright, who contributed to mice breeding and open field test; and Eloisa Pavesi, who offered valuable inputs on the manuscript. We thank Kristin Nguyen and Jessica Baker for proof-reading the manuscript. The authors declare no conflict of interest. Correspondence concerning this article should be addressed to Scott A. Heldt, Assistant Professor, Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, 855 Monroe Avenue, Memphis, TN 38163. E-mail: [email protected] 50

LACK OF NOS1 AND ADHD-LIKE BEHAVIORS

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Neale, & Faraone, 2009; Frazier-Wood et al., 2012). Behaviorally, NOS1 KO are reportedly hyperactive and display abnormal social, aggressive, and impulsive behaviors as well as deficits in learning and memory (Chiavegatto et al., 2001; Chiavegatto & Nelson, 2003; Kelley, Balda, Anderson, & Itzhak, 2009; Tanda et al., 2009; Weitzdoerfer et al., 2004). To further characterize NOS1 KO as a novel model for ADHD, we examined the behavior of these mice in tests that modeled ADHD-like symptoms in rodents, including OF, TWAA, PA, and SMAR. Furthermore, we examined whether the NO donor molsidomine (MSD) could potentially rescue ADHD-like behaviors.

Materials and Method Animals NOS1 KO (B6.129S4-Nos1tm1Plh/J, C57BL/6J congenic strain, Jackson Laboratory, Bar Harbor, ME) as described previously (Huang, Dawson, Bredt, Snyder, & Fishman, 1993) were used in this experiment. Descendants from the initial heterozygote breeding pairs were used to set up the following breeding schemes: male NOS1⫺/⫺ ⫻ female NOS1⫺/⫺, male NOS1⫺/⫺ ⫻ female NOS1⫺/⫹, and male NOS1⫹/⫹ ⫻ female NOS1⫹/⫹, to efficiently produce sibling cohorts (KO: NOS1⫺/⫺; WT: NOS1⫹/⫹; and HT: NOS1⫹/⫺) on C57BL/6 background for subsequent experiments. Animals were housed in microisolation cages with ad libitum

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access to food (Teklad rodent diet, Harlan Laboratories, Indianapolis, IN) and water. The animal facility also provided a 12 hour light:dark cycle and controlled temperature/humidity. Both males and females (in random estrous cycle) between the age of 7 weeks and 14 weeks were used for experimentation. All procedures, except overnight measurement of spontaneous activity, were carried out during the light phase of the day (900 to 1800 hrs). All protocols were approved by the Animal Care and Use Committee of the University of Tennessee Health Science Center in accord with principles and standards of animal care outlined by the National Institute of Health Institutional Animal Care and Use Committee. The assignment of animals to each experiment is described in Supplementary Material (Figure S5). In the first experiment, we found that the OF behaviors of heterozygote mice (HT) were indistinguishable from wildtype (WT) mice (see Figure 1); thus, we decided to use only WT and KO in subsequent experiments to reduce the complexity of data analysis.

Behavioral Experiment Protocols The following paradigms were selected to assess ADHD-like behaviors based on a previous work that established a novel hypothyroidism model in rodents that mimics the core clinical features of ADHD (Negishi et al., 2005). OF activity. The OF is a standard test for assessing the locomotor activity and anxiety-like behaviors in rodents (Prut &

Figure 1. Open field (OF) test indicated that nitric oxide synthase knockout mice (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: ⴱ p ⬍ .05, one-way ANOVA followed by post hoc Bonferroni test. Sample size: n ⫽ 11 for wild type (WT) (5 male and 6 female); n ⫽ 14 for HT (6 male and 8 female); n ⫽ 21 for KO (9 male and 12 female).

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Belzung, 2003; Walsh & Cummins, 1976). Tests were performed in a square OF apparatus measuring 43.2 cm W ⫻ 43.2 cm D ⫻ 30.5 cm H (Med Associates, St. Albans, VT). The animal was placed in the apparatus and allowed to explore freely for 30 min. Movement of the animal was tracked by photobeam sensors. The center was defined as the central 21.6 ⫻ 21.6 cm2. The ambulatory time, distance traveled, ambulatory activity counts, center time, stereotypic movements, and vertical rearing were analyzed by a computerized system. The results were binned to six consecutive blocks, each block lasting 5 min, for data presentation and analysis. TWAA paradigm. TWAA is a complex task that requires mice to both learn the association of the cue with shock punishment (fear learning) and acquire a strategy to escape (performance) in rodents (Darvas, Fadok, & Palmiter, 2011; Mowrer & Lamoreaux, 1946). Training and testing were conducted in shuttle boxes measuring 35.6 cm W ⫻ 17.8 cm D ⫻ 30.5 cm H (Coulbourn Instruments, Whitehall, PA), with the following modifications: a loudspeaker was mounted to the left side; the transparent plastic wall of the right side was darkened to achieve the same appearance and lighting profile as the dark side (left side); and 2 dim bulbs and 1 bright bulb were mounted to the ceiling of the chamber to provide lighting during tests. Here, the bright bulb was kept off and the 2 dim bulbs were turned on to provide ambient lighting of 2.5 lux for each side of the chamber. Over four consecutive days (Days 1– 4), mice went through TWAA training. Intraperitoneal injections of MSD (20 mg/kg, dissolved in 0.9% saline) or Sal (0.9% saline) were administered 30 min before each session. The dosage was selected based on a previous study showing that MSD (20 mg/kg) rescued contextual fear and long-term olfactory fear learning deficits in NOS1 KO and differentially regulated cyclic AMP response element binding protein (CREB) phosphorylation in KO and WT (Kelley, Anderson, Altmann, & Itzhak, 2011; Pavesi, Heldt, & Fletcher, 2013). This treatment assignment divided the subjects into eight distinct groups, namely male-WT-Sal, male-WT-MSD, male-KO-Sal, male-KO-MSD, female-WT-Sal, female-WT-MSD, female-KO-Sal, and female-KO-MSD. In each training session, mice were initially given a 2-min exploration and acclimation period followed by 100 training trials. During each trial, a 2.5-kHz, 80-dB tone conditioned stimulus (CS) was presented for 5 s or until the mouse “escaped” to the other side. If the mouse failed to escape within 5 s, a 0.3-mA footshock unconditioned stimulus (US) was delivered in accompany with the CS for an additional 2 s or until the mouse succeeded to escape. Each trial was separated by an intertrial interval (ITI) of 15 s. The following behaviors in each session were recorded and used in statistical analysis: the percentage of successful active avoidance responses during tone presentation (TWAA Response, a measurement of active avoidance performance), the percentage of failures to escape when US was present (Escape Failure, a measurement of escape behavior), the numbers of the shuttle crossings events during the initial 2 min exploration and acclimation period (EXP Crossing, a measurement of baseline activity), and the numbers of the crossing events during ITI (ITI Crossing, a measurement of the suppression of unnecessary movement and the focus on the upcoming tone). Initial analyses of dependent measures were performed using a four-way repeated-measure analysis of variances (ANOVAs) with genotype (WT, KO), sex (male, female), and treatment (MSD, Sal) as between-subjects factors and session (Day 1– 4) as within-

subjects factor. Because multiple interactions involving sex were identified as significant, males and females were subsequently analyzed separately using lower level three-way ANOVAs. Both four-way and three-way ANOVA statistical tables are presented in Supplementary Material (Tables S1.1 to S1.3). To further evaluate group differences, statistical analyses were performed by one-way ANOVA with Dunnett’s t tests for pairwise comparisons against the WT-Sal group on each testing days. PA paradigm. Not only is PA task commonly used to assess learning and memory function by the subject’s avoidance of going into a compartment previously paired with a shock (Bammer, 1982), but it is also used as a measurement of impulsivity in rodents (Marusich, Darna, Charnigo, Dwoskin, & Bardo, 2011; Matzel, Babiarz, Townsend, Grossman, & Grumet, 2008). PA training and testing sessions were conducted in the shuttle box cages described above. During these sessions, the plastic wall was uncovered and the bright bulb was turned on to deliver 150 lux illumination for the bright, transparent side. Both dim bulbs were turned off. This configuration separated the chamber into light and dark compartments. On Day 0 (training), each mouse was placed in the light compartment. After 30 s of acclimation, the guillotine door separating the compartments was lifted. After the animal entered the dark compartment, the door was closed, and three footshocks (0.6 mA, 2-s duration, separated by 10-s interval) were delivered. The animal was removed from the dark compartment 1 min after the last shock. On Days 1–3 (testing of avoidance retention), each animal was placed in the light side and allowed to enter the dark side after 30 s, when the guillotine door was opened. The step-through latency was recorded for both the training and the testing sessions and the cut-off time was set at 300 s. Spontaneous motor activity rhythms (SMAR). SMAR is a measurement developed in the last decade to assess animals’ spontaneous rhythmic motor activity in a stress-free environment (Negishi et al., 2005). SMAR was measured in conditioning chambers (30.5 cm W ⫻ 25.4 cm D ⫻ 25.4 cm H, Med Associates, St. Albans, VT) with the following modifications: a red light was mounted in each chamber to provide illumination for recording during the dark cycle, and the chamber was padded with regular bedding chips to mimic home-cage environment. Animals were provided with ad libitum access to food and water. The experiments started at 1030 hrs and the testing room was left undisturbed afterward. Two types of lighting conditions were used. For the light phase (1030 –1830 hrs), the ceiling light and the red lights were both turned on. For the dark phase (1830 – 0830 hrs next day), only the red lights were left on to provide lighting for camera recording. Activities were recorded by a digital camera at a frame rate of 3.75 fps and processed by FreezeFrame3 (Coulbourn Instruments, Whitehall, PA). The data for analysis was collected during the dark period from 2000 to 0800 hrs next day and hence started 9.5 hr after the animal was introduced to the chamber. Activity indexes generated by FreezeFrame3 were exported to Matlab (MathWorks, Natick, MA) and subjected to Fast-Fourier Transformation (FFT). The activity indices and the FFT output were normalized and smoothed using 500-point and 3-point moving window averaging algorithm, respectively. The peak frequency was defined as the frequency with the highest amplitude in the slow-wave band (0.25h⫺1–2.5h⫺1). The percentage of active time was defined as the percentage of time the animal spent during

LACK OF NOS1 AND ADHD-LIKE BEHAVIORS

which the raw activity index value was higher than 20, a threshold empirically identified to detect active movements.

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found between KO and WT and between KO and HT in all locomotor-related parameters whereas no significant difference (p ⬎ .1) was detected on anxiety-related measures.

Results

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NOS1 KO Were Hyperactive, But Not Overly Anxious, in OF Test Consistent with previous findings (Tanda et al., 2009), NOS1 KO displayed increased locomotor activity in the OF. A three-way repeated measure ANOVA with genotype (WT, HT, and KO) and sex (male, female) as between-subjects factors and block (1– 6) as with within-subject factor revealed a significant main effect of genotype on ambulatory time, distance traveled, and ambulatory activity counts (Fs(2, 40) ⬎ 3.99, ps ⬍ .05; Figure 1a– c). Effects of sex (Fs(1, 40) ⬍ 0.04, ps ⬎ .1) and Genotype ⫻ Sex interaction (Fs(2, 40) ⬍ 0.14, ps ⬎ .1) were not significant, thus males and females were grouped and plotted together. No significant difference (Fs(2, 40) ⬍ 1.36, ps ⬎ .1) was detected on the anxietyrelated scores (i.e., center time, stereotypic, or vertical activities; Figure 1 d–f). The cumulative scores were analyzed by one-way ANOVA with genotype as the independent variable followed by post hoc Bonferroni test. Significant (p ⬍ .05) differences were

Male NOS1 KO Displayed Abnormal TWAA Performance Overall, male KO displayed impairments in TWAA performance, whereas female KO generally showed no deficits. Male KO showed significant deficits in active CS avoidance response (TWAA Response, a measurement of active avoidance performance). They also displayed higher incidences of shuttle crossings during the exploratory period (EXP Crossing, a measurement of activity), intertrial interval crossings (ITI Crossing, a measurement of restlessness and inattention) and failed escape attempts (Escape Failure, a measurement of escape behavior). In male KO, MSD administered acutely before testing rescued deficits in TWAA Response and escape behavior, but did not normalize EXP Crossing, ITI Crossing. TWAA response. As seen in Figure 2a and 2e, both males and females displayed general increases in TWAA Responses across days, as revealed by significant main effects of session (F(3, 81) ⫽ 47.58 for male and F(2.16, 58.41) ⫽ 41.44 for female, ps ⬍

Figure 2. Nitric oxide synthase knockout mice (NOS1 KO), notably males, showed abnormal performances when compared with wildtype (WT) in 2-way active avoidance paradigm (TWAA), namely the percentage of successful TWAA Response to tone (a, e), the percentage of failure to escape the footshock (Escape Failure, b, f), number of exploratory period (EXP) Crossing (c, g), and number of intertrial interval (ITI) Crossing (d, h). Males and females are plotted separately. Molsidomine (MSD) (20 mg/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: ⴱ 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).

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.001, Greenhouse-Geisser correction applied) in three-way ANOVA. For males, three-way ANOVA analysis yielded significant interactions between Genotype ⫻ Treatment, Session ⫻ Treatment, and Session ⫻ Genotype ⫻ Treatment (ps ⬍ .01). One-way ANOVA with post hoc Dunnett’s t tests for pairwise comparisons against the WT-Sal revealed significant group differences (Fs(3, 30) ⬎ 5.70, ps ⬍ .01) on Day 2– 4 as seen in Figure 2a. KO-Sal demonstrated lower TWAA Response compared with WT-Sal on all test days (ps ⬍ .05). Such deficit in KO was rescued by MSD treatment as indicated by lack of statistical difference between KO-MSD and WT-Sal group (ps ⬎ .1). Of interest to the authors, MSD treatment paradoxically reduced TWAA Response in WT on Day 3 (p ⬍ .05) and Day 4 (p ⫽ .051). For females, three-way ANOVA revealed no significant main effects or interactions other than session (Supplementary Material Table S1.3), indicating that the avoidance behavior in female KO was comparable to WT; and that MSD had little effect in neither WT nor KO (Figure 2e). Together, these analyses indicate avoidance behavior deficits in male KO, which is rescued by MSD treatment; and showed a paradoxical impairing effect of MSD in male WT in later sessions. Escape failure. For males, three-way ANOVA analysis yielded significant Genotype ⫻ Treatment and Session ⫻ Genotype interactions as well as a main effect of genotype (ps ⬍ .05; Supplementary Material Table S1.2). One-way ANOVA with post hoc Dunnett’s t tests for pairwise comparisons against the WT-Sal revealed significant group differences on each testing day (Fs(3, 27) ⬎ 6.80, ps ⬍ .001), as seen in Figure 2b. KO-Sal showed higher Escape Failure on each testing day (ps ⬍ .001). This deficit in KO was rescued by MSD treatment as indicated by lack of statistical difference between the KO-MSD and WT-Sal group (ps ⬎ .1). Of interest to the authors, MSD treatment unexpectedly increased Escape Failures in WT on Day 3– 4 (ps ⬍ .05). For females, three-way ANOVA indicated no significant effects (Supplementary Material Table S1.3), indicating that the escape behavior in female KO was comparable to WT; and that MSD had little effect in neither WT nor KO (Figure 2f). Together, these analyses indicate escape behavior deficits in male KO, which could be rescued by MSD treatment; and showed a paradoxical impairing effect of MSD in male WT in later sessions. EXP crossing. One-way ANOVA with post hoc Dunnett’s t tests for pairwise comparisons against the WT-Sal revealed significant group differences on Day 3 (F(3, 27) ⫽ 4.96, p ⬍ .05) and borderline-insignificant difference on Day 4 (F(3, 27) ⫽ 2.70, p ⫽ .06) as seen in Figure 2c. KO-Sal had higher EXP Crossing on Day 3 (p ⬍ .05) when compared with WT-Sal. Such abnormality was not rescued by MSD treatment as indicated by significant differences between the KO-MSD and WT-Sal group (ps ⬍ .05) on Days 3– 4. Of interest to the authors, MSD treatment paradoxically increased EXP Crossing in WT on Day 3 (p ⬍ .05). For females, a three-way ANOVA indicated a significant effect of genotype (p ⬍ .05; Supplementary Material Table S1.3). A one-way ANOVA yielded no significant results (Figure 2g). Together, these analyses demonstrate hyperactivity in male KO in later sessions, which could not be rescued by MSD treatment; and showed a paradoxical effect of MSD in male WT. ITI crossing. One-way ANOVA with post hoc Dunnett’s t tests for pairwise comparisons against the WT-Sal revealed group differences on each testing day (Fs(3, 27) ⬎ 3.17, ps ⬍ .05) as

seen in Figure 2d. KO-Sal group exhibited higher ITI Crossing when compared with WT-Sal on each testing day (ps ⬍ .05). Such abnormality was not rescued by MSD treatment as indicated by differences between KO-MSD and WT-Sal group on Day 3 (p ⫽ .055) and Day 4 (p ⬍ .001). Of interest to the authors, MSD treatment paradoxically increased ITI Crossing in WT on Day 2 (p ⬍ .05), and such trend (although not statically significant) was observed on Days 3– 4 also. For females, the three-way ANOVA yielded a significant main effect of genotype and reliable Session ⫻ Genotype and Session ⫻ Treatment interactions (ps ⬍ .05). One-way ANOVA revealed group differences only on Day 4 (F(3, 27) ⫽ 6.68, p ⬍ .01). Post hoc Dunnett’s t tests revealed that the difference was mostly because of the KO-MSD group on that day (Figure 2h). Of note, for the WT-Sal group of either sex, the ITI Crossing score dropped and maintained at a very low level after the first day of training, suggesting that they minimized nonessential movements during ITI and were highly attentive toward the up-coming CS. Together, these analyses indicated behavioral abnormalities in KO characterized by sustained restlessness and lack of attention toward the up-coming CS during ITI, which could not be rescued by MSD treatment; and showed a paradoxical effect of MSD in male WT.

NOS1 KO Displayed Deficits in PA Acquisition and Retention Next, we investigated whether lack of NOS1 had any impact on PA acquisition and retention (see Figure 3). On the training day

Figure 3. Nitric oxide synthase knockout mice (NOS1 KO) showed deficits in passive retention test. NOS1 KO exhibited shorter step-through latency in retention tests as compared with their wildtype (WT) counterparts. Data represent means ⫾ SEM. Significance level: ⴱ p ⬍ .05; ⴱⴱ p ⬍ .01; ⴱⴱⴱ 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).

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(Day 0), both WT and KO entered the dark chamber within a short latency. Two-way ANOVA with genotype (WT, KO) and sex (male, female) as independent factors revealed no significant effect of genotype or sex (Fs(1, 39) ⬍ 2.04, ps ⬎ .1) on the latency of Day 0, although a borderline significant Genotype ⫻ Sex interaction was found (F(1, 39) ⫽ 4.37, p ⫽ .043). A three-way repeated measure ANOVA with genotype and sex as between-subjects factors and session (Day 1–3) as withinsubject factor was then performed on step-through latency of testing days. This analysis yielded significant effects of genotype (F(1, 39) ⫽ 12.29, p ⬍ .01) and session (F(1.64, 63.88) ⫽ 10.75, p ⬍ .001, Greenhouse-Geisser correction applied), but no significant effect of sex or Genotype ⫻ Sex interaction was identified (Fs(1, 39) ⬍ 0.14, ps ⬎ .1); thus, males and females were grouped and plotted together. Both KO and WT showed significant passive avoidance response on Day 1 when compared with Day 0 (ps ⬍ .01, paired t test), indicating mice of both genotypes were able to associate entering the dark compartment with the shock punishment. However, the latency was significantly shorter in KO than WT on each testing day (ps ⬍ .05, unpaired t test). On Day 3, WT still retained significant avoidance (p ⬍ .001, paired t test). However, the latency of KO already reached statistical insignificance as compared with Day 0 (p ⬎ .05, paired t test).

NOS1 KO Showed Abnormal SMAR WT typically showed four to five clusters of activity during the recording period from 2000 to 0800 hrs, which could often be directly observed from the plot of activity index. FFT revealed that the spontaneous activities in WT had a typical slow-wave rhythm with a peak frequency at ⬃0.5h⫺1, which was either disrupted or shifted to higher frequencies in KO (Figure 4a– c). Two-way ANOVA with genotype (WT, KO) and sex (male, female) as independent factors revealed a significant effect of genotype on peak frequency (F(1, 24) ⫽ 11.04, p ⬍ .01). No significant effect of sex or Genotype ⫻ Sex interaction (Fs(1, 24) ⬍ 1.62, ps ⬎ .1) was found, thus males and females were grouped and plotted together. The percent of active time was similar between WT and KO with no significant effect of genotype, sex, or Genotype ⫻ Sex interaction identified (Fs(1, 24) ⬍ 1.09, ps ⬎ .1). A nonparametric test (independent-samples Mann-Whitney U test) was also performed. The peak frequency of KO was significantly different from WT (p ⬍ .01), whereas no significant difference was detected on the percent of active time (p ⬎ .05; Figure 4a). These observations suggest that KO were likely to initiate more clusters of activity with shorter durations than WT.

Discussion Summary of Findings In the OF test, similar to past study (Tanda et al., 2009), KO demonstrate sustained hyperactivity throughout the session. However, no significant difference in other recorded OF behaviors (center time, stereotypic activity, and vertical activity) is detected; suggesting the hyperactivity of KO in our apparatus and paradigm is not because of excessive anxiety when facing the novel open field environment.

Figure 4. Nitric oxide synthase knockout mice (NOS1 KO) showed abnormal spontaneous motor activity rhythms (SMAR) in the dark phase of the day. (a) The peak frequency in the slow-wave band (left) and the percent of active time (right) were analyzed (b– c). Representative results of a wildtype (WT) and a KO mouse, showing the output of Fast Fourier Transformation (FFT) analysis (b) and the raw activity indexes (c). Data represent means ⫾ SEM. Significance level: ⴱⴱ 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).

In the TWAA test, our finding is sex-specific. Only male KO demonstrate remarkable abnormalities, characterized by avoidance and escape behavior deficits, sustained hyperactivity in an acquainted environment, restlessness in the ITI, and lack of attention toward the tone stimulus. On the other hand, although similar trends are often observed in females, the group differences are much smaller and the statistics are typically nonsignificant. These results indicate that the fine-tuning of NO concentration in the brain is particularly important for TWAA performance in males. Such phenomenon can be explained, at least in part, by the effect of estrogen, which is an activator of not only NOS1 but also NOS2 and NOS3 (Chambliss & Shaul, 2002; Karpuzoglu & Ahmed, 2006; Lekontseva, Chakrabarti, Jiang, Cheung, & Davidge, 2011), and may exert a compensatory, protective effect over the lack of NOS1 in the female KO. NO plays many sex-specific roles in the rodent brain. For instance, the plasticity of barrel cortex is impaired in male NOS1 KO but not females (Dachtler, Hardingham, & Fox, 2012). NO is also identified as a sex-specific factor mediating the effects of chronic mild stress on depressive behaviors (Hu et al., 2012). Our results contribute to another sex-specific effect of NOS1. Because ADHD is also recognized as a disorder with potential sex differences (Fedele, Lefler, Hartung, & Canu, 2012; Waddell & McCarthy, 2012), the abovementioned sex dif-

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ferences and their correlation with ADHD should be further investigated. We also find a paradoxical effect of MSD treatment in TWAA. MSD promotes TWAA behavior in male KO, whereas it elicits opposite effects in male WT. MSD is ineffective in reducing hyperactivity, restlessness, and inattention in KO. Paradoxically, MSD increases those scores in male WT. Such phenomena are especially difficult to interpret. One explanation is that the lack of NOS1 in the earlier developmental stage might cause irreversible deficits in neuronal circuits that cannot be rescued by acute administration of NO donor. Acute versus chronic treatment and the dose level might also contribute to the observed phenomena. Future studies are needed to address whether chronic and/or early treatment of NO donor in NOS1 KO can rescue these particular behavioral deficits. Additionally, another hypothesis is that NO level and the performance in TWAA follow an inverted-U function (Supplementary Material Figure S6); any deviation from the optimal range will be detrimental. This hypothesis is coherent with a previous study showing similar paradoxical effects of MSD in CREB phosphorylation. CREB phosphorylation in the amygdala is elevated in MSD-treated WT mice and reduced in MSD-treated KO mice, although the mechanism of these opposite effects and the implication on animal behavior remain unclear (Kelley et al., 2011). Inverted-U functions are key in many aspects of ADHD. It is known that the relationship between dopamine and impulsiveness/reinforcement learning (Williams & Dayan, 2005) as well as the relationship between norepinephrine and attentional performance (Howells, Stein, & Russell, 2012) appear to follow the inverted-U function. Further studies are needed to explore the underlying mechanisms of these paradoxical phenomena and explain whether excessive NO could also lead to ADHD-like behaviors. In the PA test, KO acquire a significant level of avoidance behavior after training, although the latencies are significantly shorter than WT across tests and reach statistical insignificance on Day 3 when compared with Day 0. This suggests that KO correctly associate entering the dark chamber with punishment. Past studies showing NOS1 KO had only a minimal impairment of cued fear (Kelley et al., 2009) also support the idea that KO are able to acquire Pavlovian association. Together with TWAA, these results indicate that the avoidance behavior deficits observed in KO are not entirely because of learning impairments; certain operantrelated performance deficits (restlessness in the chamber, inability to attend to the CS presentation, and inability to suppress the urge to move to the darker, more comfortable compartment) may also be involved. Learning and performance usually work synergistically in the acquisition of a particular behavior response. Based on our finding and past research showing some learning and memory deficits in NOS1 mice, we deduce that both learning and performance deficits contribute to the observed behavioral outcome. Past studies have investigated learning behavior of NOS1 KO in several learning/memory paradigms, including Morris water maze, multiple T maze (Weitzdoerfer et al., 2004), eight-arm radial maze (Tanda et al., 2009), auditory/contextual fear (Kelley et al., 2009), and olfactory fear (Pavesi et al., 2013). However, little is known about their performance in avoidance conditioning (TWAA and PA). Although these paradigms generally fall in the category of learning and memory, they are distinctive tasks that assess differ-

ent type of behaviors (avoidance). Our TWAA/PA experiments are novel addition to the behavioral profile of NOS1 KO. Many studies have investigated TWAA and PA response in rats using NOS inhibitors and NO donors/precursors (Chien et al., 2008; Komsuoglu-Celikyurt et al., 2011; Kucukatay et al., 2009; Pitsikas et al., 2001; Utkan, Gocmez, Ozer, Gacar, & Aricioglu, 2012). Majority of these studies were carried out by administration of nonselective and putative partially selective NOS1 inhibitors and NO donors/precursors. Thus, the effects because of other isoforms of NOS (NOS2 and NOS3) cannot be ruled out. To our knowledge, only one study has reported impaired PA retention using a selective NOS1 inhibitor (N␻-Propyl-L-arginine hydrochloride) in rats (Utkan et al., 2012), while little is known about its effect on TWAA. Our study shows TWAA and PA deficits in NOS1 KO mice. Thus, it not only addressed the selectivity issue among the different NOS isoforms, but also extends the existing knowledge of the effect of NO on TWAA and PA from rats to mice. This might be of great interest for many researchers who use mice as their model animal, because TWAA and PA responses are generally more difficult to obtain in mice than in rats. In the SMAR test, KO demonstrate abnormal spontaneous behavioral patterns during the dark period, indicating significant disturbance of spontaneous behaviors. This experiment also extends a previous study in rat to mice (Negishi et al., 2005), suggesting SMAR is an insightful and reliable behavioral index to investigate rodent behavior. Past researches suggest certain involvement of NO system in the circadian rhythm. Studies reveal that administration of NO donor promotes the expression of clock gene, while inhibition of NOS results in impairment of clock gene expression and disruption of circadian rhythm (Kunieda et al., 2008; Watanabe, Hamada, Shibata, & Watanabe, 1994). On the other hand, the NOS activity in the brain is also found to have a robust diurnal rhythm (Ayers, Kapas, & Krueger, 1996). However, little evidence suggests that the distinctive NOS1 isoform plays a role in the regulation of circadian rhythm. In fact, one study reports that NOS1 KO have normal circadian rhythmicity and photic entrainment response, indicating that NOS1 might not be necessary for such behavior (Kriegsfeld et al., 1999). Together, we suggest that rather than regulating circadian rhythm, NOS1 actually controls the spontaneous activity rhythm in rodents, which has a typical frequency of 0.5h⫺1, and should not be confused with the diurnal circadian rhythm. Besides the abovementioned behavioral deficits, several other abnormalities are evident in KO. Gastrointestinal problems, such as stomach hypertrophy and watery, particularly odorous feces are often found in KO. They are also prone to develop brief episodes of spontaneous or handling-induced convulsion (Supplementary Material Movie S4). Seizure and GI problems are common comorbidities of ADHD. Studies suggest that ADHD and epilepsy are bidirectionally linked, and estimate that up to 40% of the pediatric epilepsy patients may also have ADHD whereas epilepsy risk is about twice in children with ADHD than without (Cohen et al., 2013; Salpekar & Mishra, 2014). Besides, GI problems (constipation and fecal incontinence) occur far more often in patients with ADHD than without, and the fecal composition of autism spectrum disorder patients is significantly different from normal individuals (McKeown, Hisle-Gorman, Eide, Gorman, & Nylund, 2013; Wang et al., 2012). As these features are also coincidentally observed in NOS1 KO, they provide circumstantial evidence supporting our

LACK OF NOS1 AND ADHD-LIKE BEHAVIORS

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hypothesis that NOS1 KO is a good animal model for ADHD. Furthermore, even though these abnormalities might cause behavioral disturbances to a certain degree in mice, they should be comparable with the human counterparts. Of note, previous study (Kelley et al., 2009) suggests that NOS1 KO have normal pain response. Likewise, our data demonstrate that NOS1 KO retain normal footshock reactivity (Supplementary Material Figure S3). Direct observation confirms that KO are equally capable of sensing the footshock and hearing the tone, thus deficits found in KO are not because of sensory constraints.

Other Evidence to Support the Role of NOS1 in ADHD Several studies suggest a relationship between NOS1 and ADHD in human. Genetic analysis links ADHD to chromosome 12q24.3 (Frazier-Wood et al., 2012), the neighborhood where the NOS1 gene is located. A polymorphic NOS1 ex1f-VNTR allele frequently found in ADHD patients is prominently associated with dysregulation of prefrontal cortex (Reif et al., 2009) and ventral striatal (Hoogman et al., 2011) functions. It is hypothesized to be the genetic link between restless leg syndrome and ADHD (Reif, 2010). Observations of ADHD patients reveal altered blood NO levels and NOS activity in affected individuals. Some studies claim that NO level and NOS activity (Ceylan, Sener, Bayraktar, & Kavutcu, 2010, 2012) are higher in ADHD patients, whereas others report that blood NO level is lower in patients with pure ADHD, but remains normal in ADHD group comorbid with oppositional defiant disorder (Varol Tas, Guvenir, Tas, Cakaloz, & Ormen, 2006). Rodent behavioral studies using NOS1 KO indicate increased activity, abnormal social behaviors, and impaired working and spatial memory (Tanda et al., 2009; Weitzdoerfer et al., 2004). Furthermore, NOS1 KO (Nelson et al., 1995) and inhibition of NOS1 (Demas et al., 1997) both produce elevated aggressive

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behaviors in mice as measured by intruder-resident test. In molecular studies, it is well established that NO functionally interacts with glutamatergic systems, as NOS1 activation is controlled by both ionotropic and metabotropic glutamate receptors; and glutamate release as well as glutamate receptor activity are in turn modulated by NO (Boix, Llansola, Cabrera-Pastor, & Felipo, 2011; Christopherson et al., 1999; Manzoni et al., 1992; Roenker, Gudelsky, Ahlbrand, Horn, & Richtand, 2012). The NO system is extensively involved in dopaminergic, noradrenergic, and serotonergic transmission by inhibiting monoamine transporters (Kiss & Vizi, 2001; Kiss et al., 2004), activating aromatic L-amino acid decarboxylase (Duchemin et al., 2010), and modulating dopamine release (Smith & Whitton, 2001; Zabel et al., 2002). The NO system is also influenced by GABA, acetylcholine, and neuropeptides (Fedele & Raiteri, 1999). Electrophysiological studies reveal that NO modulates striatal function through its inhibitory effect on projection neurons and excitatory effect on spontaneously active neurons (Galati et al., 2008). These findings (as summarized in Figure 5) indicate that NOS1 exerts critical modulatory effects on many neuronal signaling pathways, such as those for long-term potentiation/depression and reward circuitry; and suggest that the action is required for maintaining normal functions of the brain areas crucial for ADHD-like behaviors, such as prefrontal cortex and striatum. Although circumstantial, they could serve as the foundation to support the hypothesis that lack of NOS1 produces ADHD-like behaviors.

Validity of NOS1 KO as Rodent Model for ADHD A good animal model for ADHD should meet the following criteria: (a) face validity, in which the model should reflect the key features of the disease; (b) construct validity, in which the similarities between the model and the disease could be explained by theoretic rationales; and (c) predictive validity, in which unknown

Figure 5. Summary of evidence supporting the hypothesis that dysfunction of nitric oxide synthase and nitric oxide system may result in attention deficit hyperactivity disorder-like behaviors.

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aspects of the disease could be predicted by the model (Sagvolden, Russell, Aase, Johansen, & Farshbaf, 2005). Experimental avoidance paradigms such as TWAA and PA are used both to measure learning/memory as well as other behaviors such as impulsiveness and maladaptive behaviors. ADHD patients often exhibit decreased likelihood of deploying active coping strategies when facing a challenge and would rather prefer an immediate negative result (Knouse & Mitchell, 2014). Furthermore, they often exhibit impaired behavioral inhibition and increased rate of committing passive avoidance error (Farmer & Rucklidge, 2006). These could translate into the observed TWAA/PA deficits in mice. Our results also reveal that lack of NOS1 produces a behavioral profile characterized by sustained hyperactivity, learning impairments, potential deficits of attention, restlessness, sex differences, dysregulation of spontaneous activity rhythm, and other symptoms including gastrointestinal problems and seizure. These features coincide with many clinical signs of ADHD and suggest sufficient face validity of the model. Of note, hyperactivity, a main phenotypic deficit found in NOS1 KO, might intrinsically confound the behavioral tests and make certain parameters hard to interpret. For example, the inattention, as measured by ITI Crossing in TWAA, could only be circumstantially inferred but not directly concluded. Future experiments using more specifically designed tests such as multiple-choice serial RT task and go/no-go task (Bushnell & Strupp, 2009; Winstanley, Eagle, & Robbins, 2006) are desired to further characterize inattention and impulsiveness in NOS1 KO. The extensive interactions between NO and the monoamine system are well studied and could underpin the relationship between the genotypic deficit and the phenotypic outcome in NOS1 KO, establishing construct validity. Further, recent clinical studies supporting the linkage between NOS1 and ADHD in humans illustrate, at least in part, the predictive validity of the model. Future studies, such as investigating the effect of methylphenidate (MPD), a common clinical treatment option for ADHD, in NOS1 KO, would further test the predictive validity of this model.

2013), central nucleus of amygdala (Shen, Guo, & Yu, 2004), and cerebral cortex (Qadri et al., 2003) in SHR. On the other hand, NO activity in hippocampus is reportedly increased in rats with experimental neonatal and juvenile hypothyroidism and it is believed to contribute to learning and memory deficits (Hosseini et al., 2010). Further investigations are needed to evaluate the functional role of NOS1 as well as the efficacy of MSD treatment in those animal models of ADHD. While MPD is a treatment option for ADHD, recently studies have identified potential risks associated with its use and misuse, which opens the door for alternative treatments. (Brookshire & Jones, 2012; Carlezon, Mague, & Andersen, 2003; Godfrey, 2009; Sadasivan et al., 2012). As previously mentioned, NOS1 ex1fVNTR polymorphism, which reduces NOS1 expression, is frequently identified in ADHD patients; there is a rationale that MSD (and potentially other NO donors) could be a possible target for evaluation as a novel personalized therapeutic approach for ADHD patients with such genetic traits who suffer from learning disabilities. MSD, an orally active NO donor, was previously studied in treating stable angina and atherosclerosis and was well-tolerated by patients (Messin, Dubois, & Famaey, 2008; Van Hove, CarreerBruhwyler, Geczy, & Herman, 2005). With its safety profile already understood in humans, further testing of this compound in other animal models of ADHD and, if found effective, in human patients, should be worth pursuing. In summary, we present evidence of hyperactivity, learning impairments, restlessness, inattention, sex differences, and disturbance of rhythmic behaviors in NOS1 KO. These findings expand the existing knowledge of the behavioral abnormalities of NOS1 KO, extend the clinical-genetic studies of NOS1s role in ADHD to a rodent model, support the hypothesis that dysfunction of NOS1 could lead to ADHD-like behaviors, and may facilitate new venues of research in ADHD neuropathology and treatment in the future.

Future Outlook and Conclusion

American Psychiatric Association. (2000). Diagnostic and Statistical Manual of Mental Disorders (4th ed., text rev.). Washington, DC: American Psychiatric Association. Ayers, N. A., Kapás, L., & Krueger, J. M. (1996). Circadian variation of nitric oxide synthase activity and cytosolic protein levels in rat brain. Brain Research, 707, 127–130. http://dx.doi.org/10.1016/00068993(95)01362-8 Bammer, G. (1982). Pharmacological investigations of neurotransmitter involvement in passive avoidance responding: A review and some new results. Neuroscience and Biobehavioral Reviews, 6, 247–296. http://dx .doi.org/10.1016/0149-7634(82)90041-0 Boix, J., Llansola, M., Cabrera-Pastor, A., & Felipo, V. (2011). Metabotropic glutamate receptor 5 modulates the nitric oxide-cGMP pathway in cerebellum in vivo through activation of AMPA receptors. Neurochemistry International, 58, 599 – 604. http://dx.doi.org/10.1016/j.neuint .2011.01.025 Brookshire, B. R., & Jones, S. R. (2012). Chronic methylphenidate administration in mice produces depressive-like behaviors and altered responses to fluoxetine. [Advance online publication]. Synapse, 66, 844 – 847. http://dx.doi.org/10.1002/syn.21569 Bushnell, P. J., & Strupp, B. J. (2009). Assessing attention in rodents. In J. J. Buccafusco (Ed.), Methods of behavior analysis in neuroscience (2nd ed., chapter 7). Boca Raton, FL: CRC Press. Cak, H. T., Dinc, G. S., Tuzun, Z., Evinc, S. G., Cop, E., & Cuhadaroglu Cetin, F. (2013). Comorbidity and continuity of attention deficit hyper-

As previously mentioned, follow-up experiments addressing MPD’s ability to normalize the observed behavioral deficits in NOS1 KO are of great importance. Previous studies report that long-term administration of methylphenidate in adolescent rats significantly increases NOS1 expression in the striatum, an important brain region in reward circuitry (Cavaliere et al., 2012). However, the functional role of NOS1 in methylphenidate treatment is largely unclear and remains an intriguing venue for future investigation. On the other hand, it is debatable whether negative reactivity toward methylphenidate treatment precludes the use of an animal model in ADHD studies. For example, the spontaneously hypertensive rat (SHR), the prevailing rodent model of ADHD, is largely insensitive toward MPD treatment in paradigms such as open field, differential reinforcement of low-rate responding task, and five-choice serial RT test (van den Bergh et al., 2006). It is also interesting to address whether NOS1 function is abnormal in other animal models of ADHD and whether NO donor treatment has any effects in those models. Studies assert that NOS1 expression and/or activity is reduced in the brain stem (Hojna, Kunes, & Zicha, 2010), nucleus tractus solitarius (Murphy et al.,

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Received July 1, 2014 Revision received November 4, 2014 Accepted November 21, 2014 䡲