Neonatal 6-OHDA lesion model in mouse induces Attention-Deficit/ Hyperactivity Disorder (ADHD)-like behaviour

Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder characterized by impaired attention, impulsivity and hyperactivity. The “neonatal 6-hydroxydopamine” (6-OHDA) lesion is a commonly used model of ADHD in rat. However, a comprehensive assessment of ADHD‐like symptoms is still missing, and data in mouse remain largely unavailable. Our aim was to analyse symptoms of ADHD in the mouse neonatal 6‐OHDA model. 6‐OHDA mice exhibited the major ADHD‐like symptoms, i.e. hyperactivity (open field), attention deficit and impulsivity (five‐choice serial reaction time task). Further, the model revealed discrete co‐existing symptoms, i.e. anxiety‐like (elevated plus maze test) and antisocial (social interaction) behaviours and decreased cognitive functioning (novel object recognition). The efficacy of methylphenidate, a classical psychostimulant used in the treatment of ADHD, was also evaluated. A histological analysis further supports the model validity by indicating dopamine depletion, changes in cortical thickness and abnormalities in anterior cingulate cortex neurons. A principal component analysis of the behaviour profile confirms that the 6‐OHDA mouse model displayed good face and predictive validity. We conclude that neonatal dopamine depletion results in behavioural and morphological changes similar to those seen in patients and therefore could be used as a model for studying ADHD pathophysiological mechanisms and identifying therapeutic targets.

Since the dorsal anterior cingulate cortex (ACd) is one of the main targets involved in the modulation of attention and executive functions, we investigated the morphology of layer II-III pyramidal neurons of the ACd using Golgi staining (Fig. 1D1). Qualitative abnormalities of dendritic branching were noted in various regions of Golgi-Cox stained cortical tissue from 6-OHDA mice. Although not quantified, observations at high magnification indicated a lower complexity in branching of medium-sized ACd neurons (Fig. 1D1), and an apparent decrease in the size of dendritic spines (Fig. 1D1). The mean spine density on the apical (A) and basal (B) dendrites of layer III ACd pyramidal neurons is significantly reduced in 6-OHDA animals as compared to shams ( Fig. 1D2; n = 10 neurons/animal, n = 3 mice/group; in total n = 30 neurons/group; p < 0.05). In 6-OHDA mice, the area of pyramidal neurons is smaller than in sham ( Fig. 1D3; p < 0.05). Moreover, the diameter of apical dendrites of the layer II-III pyramidal neurons at 10 μm (proximal) and 100 μm (distal) from the soma showed a significant reduction in 6-OHDA mice at both distance ( Fig. 1D4-5; p < 0.05).
Impairment of short-term memory in 6-OHDA adolescent mice. In the present study, we assessed also cognitive functions through memory and learning. Object recognition was ascertained by greater time interacting with the novel than the familiar object (assessed by the ratio of time spent, Tr), and a discrimination ratio (Dr) above 0.5 (see suppl. methods) (Fig. 3J,K).
Interestingly, we found that 6-OHDA adolescent mice showed an increase in anxiety, antisocial and aggressive behaviors, and deficits in learning and memory system, which are the discrete symptoms of ADHD. In addition, Mph improved cognitive malfunctions, enhances social interactions, but has no effect in anxiety-like behavior.  (3,27) = 6.94, p > 0.05; respectively) had no effect. During the acquisition phases of the last 4 sessions of training at 1 s of SD, 6-OHDA mice were less accurate than sham mice (t = 6.22, p < 0.001; Fig. S2A), made more omission (t = 8.00, p < 0.001; Fig. S2B), premature (t = 5.10, p < 0.001; Fig. S2C) and perseverative responses (t = 6.19, p < 0.001; Fig. S2D). Thus, our data suggest that attentiveness is impaired and impulsivity increased in 6-OHDA mice.
To test that possibility, we manipulated the inter-trial interval (ITI) and the stimulus duration (SD) to evaluate impulsivity and attention, respectively, when mouse performances remained stable. Impulsivity test. Two-way ANOVA repeated measures with lesion and (ITI) as main factors, showed a significant effect of lesion (F (1, 18) = 45.64, p < 0.001), while the ITI and the interaction lesion/ITI had no effect on accuracy In contrast, the interaction lesion/ITI had no effect on those parameters (F (2,18) = 1.27; F (2,18) = 1.42; F (2,18) = 1.39, p > 0.05; respectively). When the ITI was lengthened from 5 to 7 or 10 sec, a significant increase in premature (t = 2.40, p < 0.05; t = 3.38, p < 0.01; respectively), perseverative (t = 2.03, p < 0.05; t = 3.30, p < 0.01; respectively), and omissions (t = 2.30, p < 0.05; t = 3.46, p < 0.01; respectively) responses was observed in 6-OHDA mice (Fig. 4A). This suggests that 6-OHDA mice impulsivity was disclosed by ITI increase. Meanwhile, we did not observe difference on accuracy in 6-OHDA mice when the ITI increased (t = 0.07 or t = 0.14, p > 0.05; Fig. 4A). No change in these parameters was observed in the sham group.   (see table S1 and Fig. 5A, Comp 2 in bold). Control, sham and 6-OHDA are not clearly separated along this axis (Fig. 5B). This indicated that the 6-OHDA mouse model is a good model to analyze ADHD symptoms related to the variables that composed the x-axis i.e aggression, hyperactivity and impulsivity (Fig. 5B). Mph significantly reduced the symptoms along the x-axis, but not the y-axis (Figs 5B, S3B1 and B2). Finally, in control groups,  Mph has opposite effects, significantly modifying group values along the y-axis ( Fig. S3C1 and C2, p < 0.001, Monte-Carlo). Therefore, Mph may favour anxiety and memory impairments in control.

Discussion
The diagnostic of ADHD, like other psychiatric disorders, relies on behavioural assessment. Animal models of ADHD must mimic clinical symptomatology and in particular the three core symptoms of hyperactivity, impulsivity, and impaired attention 8,14 , but also other comorbid affections.
Here we provide evidence that the 6-OHDA mouse model exhibits known major symptoms of the human pathology, namely hyperactivity in a novel environment at a juvenile stage, inattention and impulsive-like behavior at adulthood. Moreover, the 6-OHDA adolescent mouse exhibits co-morbid symptoms including increased anxiety, antisocial and aggressive behaviors, and deficits in learning and memory. We also discuss below the mechanisms underlying dopamine depletion-induced pathophysiology and we point to the interest of the 6-OHDA model in mimicking the effects of known treatments.
The 6-OHDA mouse model exhibits known symptoms of the human pathology. Disrupting brain systems through neonatal 6-OHDA lesion is a classical neurodevelopmental model of ADHD in rat 11,15 . Selective removal of DA projections to forebrain in neonatal rats leads to age-limited spontaneous motor hyperactivity [16][17][18] at an age corresponding to human periadolescence 11,19 . Only one study used such a model in mouse and reproduced similar locomotor impairment 12 . However, data regarding impulsive behavior or attention deficits remain unavailable in mouse 13 , or contradictory in rat 10,15,20 . Moreover, existing studies do not describe discrete co-morbid symptoms.
ADHD adult patients show inattention and elevated impulsivity 21 that can be illustrated by ADHD patient performance of the continuous performance task 22,23 . ADHD subjects have slower and more variable reaction times, and make more errors of omission indicative of poor attentional ability 21 . In addition, they make more errors of commission, demonstrating reduced behavioral inhibition and impulsivity. In our study, hyperactivity is determined in 6-OHDA mice with the open field test. One of our main findings was that 6-OHDA adult mice displayed deficits in inhibitory control in the 5-CSRTT, a task used in rodents that requires behavioral inhibition 24 . 6-OHDA mice exhibit an increased number of perseverative responding under baseline conditions and increased premature responding during the inter-trial interval challenge. Impulsive choice reflects, to a greater degree, decision-making processes rather than motoric inhibition 25 . This is generally considered to reflect a failure of the «executive system» represented by frontal cortical areas exerting a top-down control to limbic and paralimbic areas 26 .
Likewise, 6-OHDA mice displayed a greater loss of accuracy when attention was challenged. Interestingly, this effect was present all along the session, indicative of a deficit in selective attention and difficulty to maintain sustained attention. Taken together, these data demonstrate that 6-OHDA adult mice exhibit attention deficit and impulsivity.
Beside the major symptoms, ADHD children exhibit cognitive impairments and short-term memory deficits 5,27,28 . Access to novelty (e.g. object or environment) can elicit approach behaviors in rodents. Starting from this observation 29 , a new behavioral test was developed in the late 1980s: the so-called object recognition test 30 . The test is based on the rodent tendency to interact more with a novel than a familiar object. The exploration time of a new object during the test trial was not significantly increased in 6-OHDA mice, suggesting that cognitive abilities (e.g. learning) and/or recognition memory were impaired in 6-OHDA adolescent mice. These findings are in agreement with previous studies showing cognitive impairments in spatial discrimination task in rats 10 and in mice 12 . Anxiety disorder is also a common comorbidity of ADHD 27 that we explored with the EPM test. 6-OHDA adolescent mice exhibited anxiety-like behavior in agreement with previous results 12 . Another set of discrete symptoms of ADHD is characterized by aggression and disruptive behavior [31][32][33][34] . We found that 6-OHDA adolescent mouse showed an antisocial behavior, including reduced social interaction and aggressive attitude.
Differences with other existing animal models of ADHD. Very few animal models of ADHD have been able to mimic multiple deficits at the same time. In the early 1960s, the spontaneously hypertensive rat (SHR) was developed by inbreeding Wistar-Kyoto (WKY) rats 35 . SHR shows several major ADHD-like symptoms such as hyperactivity 36 , impulsivity and poor attention 37 . However, SHR rats also show hypertension that is not reported in patients with ADHD 14 , thus making difficult to disassociate the effects of the two disorders. Indeed, hypertension is a potential confounding factor for SHR as a model for ADHD, suggesting that altered norepinephrine (NE) transmission 38,39 may contribute to hypertension rather than hyperactivity 10 . Moreover, the WKY control often shows low activity levels, and has even been suggested as a model of depression [40][41][42] .
Another genetic model is the mouse strain lacking the dopamine transporter (DAT KO) 43 . DAT KO mice show hyperlocomotion in novel environment 43 and impaired learning and memory 44 . However, DAT KO mice display extremely elevated dopamine levels in the striatum and nucleus accumbens 45 unlike ADHD patients 46,47 . Moreover, testing this model predictive validity with psychostimulants is impossible because of the absence of the DAT protein, the primary target of these drugs 48 . Other mouse genetic models have been proposed but lack face and/or predictive validity, e.g. the Coloboma Mutant Mouse 49 .

Mechanisms of dopamine depletion-induced pathology. Hyperactivity in human subjects with
ADHD is accompanied by decreased dopamine in striatum, prefrontal cortex, septum, midbrain and amygdala 46,50,51 . Furthermore, multiple lines of evidence recently supported the view that neuroanatomical alterations exist in ADHD patients. Studies have reported decreased brain volume in patients with ADHD, slowed maturation and reduced connectivity in the prefrontal cortex, anterior cingulate cortex, basal ganglia, and cerebellum 13 . In particular, the dorsal cingulate cortex, which plays a key role in the modulation of attention and executive functions 52 , appears to be dysfunctional in patients with ADHD 4 . Moreover, lateral prefrontal development in children with ADHD is delayed by several years 53 , and the anterior cingulate cortex is thinner in adults with Scientific RepoRts | (2018) 8:15349 | DOI:10.1038/s41598-018-33778-0 ADHD 54 . Studies using functional magnetic resonance imaging in ADHD children sitting still or performing a continuous task also showed smaller sizes of DA target areas, including the prefrontal cortex and striatum and deficits in the basal ganglia 55 . Our data demonstrate that 6-OHDA mouse model of ADHD also exhibits such anatomical characteristics of ADHD, including decreased in brain weight and volume brain, smaller sizes of DA target areas, cortical thickness and abnormalities in dorsal anterior cingulate neurons.
Predictive validity of the 6-OHDA mouse model. Animal models of ADHD should be capable of predicting therapeutic effects in patients. Human studies have shown that Mph increases impulse control 56 , attention 57 , and working memory 58 in ADHD patients. Comparable findings have been obtained in rats 59 . We show here that therapeutic-equivalent doses of Mph effectively attenuate the ADHD-like symptoms in 6-OHDA mouse model. Mph reduces hyperactivity of 6-OHDA mice. Moreover, pre-treatment with Mph 30 min before testing 6-OHDA mice in 5-CSRTT resulted in increased attention. The PCA also confirms that Mph is most effective on both the major and discrete symptoms. In contrast, excessive DA stimulation might cause PFC dysfunction leading to impaired inhibition of undesirable behavior, and a deficit in sustaining attention 60 . This can explain the opposite effects of Mph observed in sham group. Limits of the model. Mice are nocturnal animals and displayed their main activity peak at the beginning of the dark phase. They have more locomotor activity during night time than they would during the day. However, in the literature, most protocols use a normal and non-inverse light-dark cycle. All behavioral tasks were performed here at the same period (morning or afternoon) for all animals used to avert any circadian related fluctuation in the performance of the animals. Neurochemical lesion studies have been able to assign specific roles in the regulation of behaviour to discrete brain areas, circuits and neurotransmitter systems, and to mirror the effects of dysfunctional anatomical loci as seen in humans with ADHD 61 . However, ADHD symptoms are the result of several dysfunctional loci interacting within a neural network to produce observable behaviours. Nevertheless, 6-OHDA lesion, although artificial, has comparable behavioural consequences and hence has important implications for research on the specific hypothetical construct 61 . The fact that so many of the ADHD symptoms can be simulated highlights the possibility that the syndrome may have multiple aetiologies, which however may impinge on common neural systems. These systems respond to a common pharmacological treatment, e.g. psychomotor stimulants. Hence, it is possible that the 6-OHDA mouse model reproduces the symptoms of the human disease through the dysfunction of this common target system of the pharmacological treatment.
Because psychostimulants, which increase catecholamine neurotransmission, have been the primary ADHD treatment for decades, clinicians and researchers conjectured that hypodopaminergic function is the neurobiological mechanism underlying ADHD. However, a feature of the 6-OHDA rat model of ADHD is the hyperactivity of the remaining dopaminergic system, and changes in dopamine receptor expression and function, even if the overal effect is a decrease of dopaminergic transmission [62][63][64][65] . This reveals a profound compensation and attempts to maintain homeostasis in the residual dopamine terminals in adulthood.
While both dopamine and norepinephrine are known to regulate motor activity, attention, learning, and cognition, dopamine has been the focus of ADHD research. In the neonatal 6-OHDA-lesioned rat model, selective dopamine depletion is achieved by pretreatment with desipramine, which protects noradrenergic nerves. This model illustrates that dopamine depletion alone is sufficient to produce ADHD-like behaviors 66 . Behavioral studies using noradrenergic drugs on animal models indicate that norepinephrine transmission does, indeed, affect ADHD symptoms, but the outcomes are mixed. Enhancing norepinephrine transmission by blocking the NET improves hyperactivity in neonatal 6-OHDA-lesioned rats 15 . From these studies, one cannot infer a causal relationship between increasing/decreasing norepinephrine neurotransmission and severity of ADHD symptoms. Instead, these studies suggest that norepinephrine has dual effects on ADHD-like behaviors 61 .
Currently no serotonergic medications are prescribed in the treatment of ADHD. most studies using animal models of ADHD suggest that serotonin acts to compensate for aberrant dopamine and/or norepinephrine signaling. 6-OHDA-lesioned rats exhibit serotonergic hyperinnervation and the elimination of serotonergic hyperinnervation by administration of the selective serotonergic toxin 5,7-DHT greatly potentiates hyperactivity 63 . Conversely, an increase in serotonergic transmission via serotonin agonist m-CPP or SERT blocker, fluoxetine, greatly reduces hyperactivity 67,68 .

Conclusion
Although animal models created by the use of neurotoxins do not inform about the causes of ADHD, they are useful tools for studying the contribution of specific brain areas or circuits to cognitive processes that are affected by this pathology 69 . The use of 6-OHDA animal models of ADHD can inform on the cognitive functions sub-served by the lesioned area. It aids to a better understanding of this disorder by parceling the ADHD syndrome into distinct comorbid deficits and provides a valid and suitable animal model for pharmacological tests. We demonstrated in the present study that neonatal 6-OHDA mouse model of ADHD is a valid and reliable model for pre-clinical studies on this neurodevelopmental disorder. 6-OHDA neonatal lesion at P5. Intracerebroventricular injection of 6-OHDA was performed at P5 following published protocol 12 . Pups received 6-OHDA hydrobromide (Sigma-Aldrich, France) or vehicle in one of the lateral ventricles after desipramine hydrochloride pretreatment (Sigma-Aldrich, France), under hypothermal anesthesia. The site of injection was set at 0.6 mm lateral to the medial sagittal suture, 2 mm rostral to the lambda and 1.3 mm in depth from the skull surface. 60 mice (P5) were divided on Sham and 6-OHDA groups (n = 30/ each group). Each experimental animals (sham and 6-OHDA) were divided into three sub-groups (n = 10; vehicle (saline), Mph 3.0 mg/kg and 5.0 mg/kg). After injection, 20% of the lesioned mice died before weaning, whereas 60-80% developed hyperactivity together with dopamine (DA) depletion ( Fig. 1A1-2). Mice that did not meet these 2 criteria were excluded from the data analysis.

Animals
Methylphenidate treatment for non-operant tests. Mph or vehicle was administered intraperitoneally (10 ml/kg in 0.9% NaCl) 45 min prior to test sessions for all the behavioral tests. The Mph doses were chosen based on previous studies [70][71][72] . The injection was given once before open field test, elevated plus maze test, social interaction test and then before the training day (2 nd day) of the novel object recognition test (Fig. 6A).
Histology. At the end of the Mph test, in the 5-CSRTT, the saline mice of both groups (sham and 6-OHDA, n = 10 each) were used for histology. Tissue preparation, Nissl staining, Golgi Cox staining and TH Immunohistochemistry were assessed as described previously [73][74][75] . The slides were visualized with an Olympus microscope and images were acquired with an Olympus D72 camera.

Behavioral tests.
Spontaneous activity (open field test) was assessed in all groups at P24. From P40, behavioral and cognitive deficits were tested in all groups (see Fig. 6A): anxiety-like behavior (elevated plus maze test at P40), anti-social behavior (social interaction test at P42), short-term memory impairment (novel object recognition test at P45-46) [76][77][78] . For the operant test, we selected only saline mice from sham and 6-OHDA groups (n = 10, each) to evaluate attention and impulsivity (5-CSRTT training from P50) 79 . As used in various studies [80][81][82][83] , and reviewed by Robbins 84 , the percentage of correct responses, also termed response accuracy, reflects errors of commission without including errors of omission and is one of the two variables best accounting for attentional performance. The percentage of omissions (no response after stimulus presentation) is the second variable accounting for attention; it reflects detection failures. The number of premature responses is an index of impulsivity. The number of perseverative responses corresponds to another form of inhibitory deficit related to impulsive/compulsive behavior.
Upon training completion in 5-CSRTT (P90), once the animals showed a stable performance in the task, the inter-trial interval (ITI) was increased (7-10 s) and the stimulus duration (SD) was decreased (0.8-0.5 s) to challenge impulsivity and attention, respectively (Fig. 6B). Each parameter was manipulated once a week during Upon training completion, once the animals showed a stable performance in the task, the inter-trial interval (ITI) was increased (7-10 s) and the stimulus duration (SD) was decreased (0.8-0.5 s) to challenge impulsivity and attention, respectively. After the behavioral challenge, mice were habituated to saline injections for 1 week. During the pharmacological challenge, Mph (3.0 and 5.0 mg/kg) was injected twice a week before the testing session. x4 or x5: number of weeks for training, impulsivity, attention, and methylphenidate tests.
Scientific RepoRts | (2018) 8:15349 | DOI:10.1038/s41598-018-33778-0 8 weeks: 1 st and 2 nd weeks, ITI = 7 s; 3 rd and 4 th weeks, ITI = 10 s; 5 th and 6 th weeks, SD = 0.8 s; and 7 th and 8 th weeks, SD = 0.5 s. After the behavioral challenge, mice were habituated to saline injections for 1 week. During the pharmacological challenge, Mph (3.0 and 5.0 mg/kg) were injected twice a week before the testing session for five weeks (Fig. 6B). 0.9% saline was injected i.p. on Tuesdays and Thursdays (baseline condition), while a given dose of Mph was administered 45 min before the session (3.0 mg/kg and 5.0 mg/kg on Wednesdays and Fridays, respectively). Mice were subjected to standard sessions of the 5-CSRTT with the same parameters used for the assessment of baseline responding. Statistical analysis. Statistical analyses were conducted using SigmaPlot 11.0 software (SigmaStat, Systat Software Inc, San Jose, CA, USA). Results were presented as mean ± standard error of the mean (SEM). The Student's t-test was used for two-sample comparisons. For multiple sample comparisons, Two-Way ANOVA was performed, followed by Student-Newman-Keuls post-tests for behavioral analysis. Two-Way Repeated measure ANOVA followed by Holm-sidak Method post-tests was used to analyze the performance in the 5-CSRTT during baseline, ITI and SD manipulations. For the pharmacological challenges, the mean of all sessions of each 5-CSRTT parameters taken in two different sessions (vehicle and each drug dose) was used as within-subjects factor and the lesion as the between-subjects factor. A Holm-sidak Method post-tests was used to follow-up significant main effects and interactions. Principal component analysis was performed using R software (Ade4 package) based on the 20 behavioral variables that have been measured in this study 85 .