Preclinical Models of Attention Deficit Hyperactivity Disorder: Neurobiology, Drug Discovery, and Beyond.

OBJECTIVE: We offer an overview of ADHD research using mouse models of nicotine exposure. METHOD: Nicotine exposure of C57BL/6 or Swiss Webster mice occurred during prenatal period only or during the prenatal and the pre-weaning periods. Behavioral, neuroanatomical and neurotransmitter assays were used to investigate neurobiological mechanisms of ADHD and discover candidate ADHD medications. RESULTS: Our studies show that norbinaltorphimine, a selective kappa opioid receptor antagonist is a candidate novel non-stimulant ADHD treatment and that a combination of methylphenidate and naltrexone has abuse deterrent potential with therapeutic benefits for ADHD. Other studies showed transgenerational transmission of ADHD-associated behavioral traits and demonstrated that interactions between untreated ADHD and repeated mild traumatic brain injury produced behavioral traits not associated with either condition alone. CONCLUSION: Preclinical models contribute to novel insights into ADHD neurobiology and are valuable tools for drug discovery and translation to benefit humans with ADHD.

Frontal Cortical Monoamine Release, Attention, and Working Memory in a Perinatal Nicotine Exposure Mouse Model Following Kappa Opioid Receptor Antagonism.

Perinatal nicotine exposure (PNE) produces frontal cortical hypo-dopaminergic state and attention and working memory deficits consistent with neurodevelopmental disorders such as attention deficit hyperactivity disorder (ADHD). Methylphenidate alleviates ADHD symptoms by increasing extracellular dopamine and noradrenaline. Kappa opioid receptor (KOR) antagonism may be another mechanism to achieve the same results because KOR activation inhibits frontal cortical dopamine release. We administered the selective KOR antagonist norbinaltorphimine (norBNI) (20 mg/kg; intraperitoneal) or methylphenidate (0.75 mg/kg; intraperitoneal) to PNE mouse model and examined frontal cortical monoamine release, attention, and working memory. Both compounds increased dopamine and noradrenaline release but neither influenced serotonin release. Both compounds improved object-based attention and working memory in the PNE group, with norBNI's effects evident at 2.5 h and 5.5 h but absent at 24 h. Methylphenidate's effects were evident at 0.5 h but not at 2.5 h. norBNI's effects temporally coincided with frontal cortical c-Jun N-terminal kinase phosphorylation. norBNI did not alter tissue dopamine content in the nucleus accumbens, offering preliminary support for lack of reinforcement.

Transgenerational transmission of behavioral phenotypes produced by exposure of male mice to saccharin and nicotine.

The use of non-nutritive sweeteners such as saccharin is widely prevalent. Although saccharin is considered safe for human consumption, it produces behavioral changes in experimental animals. We report that saccharin's behavioral effects are much more pervasive than currently recognized. In a mouse model, saccharin exposure produced motor impulsivity not only in the saccharin-exposed males but also in their offspring. In addition, the offspring showed locomotor hyperactivity and working memory deficit not observed in fathers. Spermatazoal DNA was hypermethylated in the saccharin-exposed fathers, especially at dopamine receptor promoter regions, suggesting that epigenetic modification of germ cell DNA may mediate transgenerational transmission of behavioral phenotypes. Dopamine's role in hyperactivity was further highlighted by the finding that the stimulant drug methylphenidate mitigated the hyperactivity. Nicotine is another substance that is widely used. Its use via smokeless tobacco products, some of which contain saccharin, is on the rise contributing to concerns about adverse outcomes of co-exposure to saccharin and nicotine. We found that co-exposure of male mice to saccharin and nicotine produced significant behavioral impairment in their offspring. Thus, our data point to potential adverse neurobehavioral consequences of exposure to saccharin alone or saccharin and nicotine for the exposed individuals and their descendants.

Is Paternal Smoking at Conception a Risk for ADHD? A Controlled Study in Youth With and Without ADHD.

Objective: Based on emerging preclinical findings suggesting that paternal smoking at conception may be a risk for ADHD in the offspring, we investigated whether a similar effect can be observed in humans. Method: We analyzed data from an opportunistic dataset of girl probands with (N = 140) and without (N = 122) ADHD with available information on paternal smoking at conception. Data were analyzed using Pearson's chi-square tests and multiple logistic regression. Results: ADHD probands had a significantly higher rate of paternal smoking at conception than controls (35% vs. 23%, χ2 = 3.82, p = .05) with a significant odds ratio of 1.5. However, the association lost significance after controlling for paternal ADHD, most likely due to limited statistical power. Conclusion: While preliminary, findings suggest that paternal smoking at conception may be a risk factor for ADHD in the offspring.

Nicotine exposure of male mice produces behavioral impairment in multiple generations of descendants.

Use of tobacco products is injurious to health in men and women. However, tobacco use by pregnant women receives greater scrutiny because it can also compromise the health of future generations. More men smoke cigarettes than women. Yet the impact of nicotine use by men upon their descendants has not been as widely scrutinized. We exposed male C57BL/6 mice to nicotine (200 µg/mL in drinking water) for 12 wk and bred the mice with drug-naïve females to produce the F1 generation. Male and female F1 mice were bred with drug-naïve partners to produce the F2 generation. We analyzed spontaneous locomotor activity, working memory, attention, and reversal learning in male and female F1 and F2 mice. Both male and female F1 mice derived from the nicotine-exposed males showed significant increases in spontaneous locomotor activity and significant deficits in reversal learning. The male F1 mice also showed significant deficits in attention, brain monoamine content, and dopamine receptor mRNA expression. Examination of the F2 generation showed that male F2 mice derived from paternally nicotine-exposed female F1 mice had significant deficits in reversal learning. Analysis of epigenetic changes in the spermatozoa of the nicotine-exposed male founders (F0) showed significant changes in global DNA methylation and DNA methylation at promoter regions of the dopamine D2 receptor gene. Our findings show that nicotine exposure of male mice produces behavioral changes in multiple generations of descendants. Nicotine-induced changes in spermatozoal DNA methylation are a plausible mechanism for the transgenerational transmission of the phenotypes. These findings underscore the need to enlarge the current focus of research and public policy targeting nicotine exposure of pregnant mothers by a more equitable focus on nicotine exposure of the mother and the father.

Attention and working memory deficits in a perinatal nicotine exposure mouse model.

BACKGROUND: Cigarette smoking by pregnant women is associated with a significant increase in the risk for cognitive disorders in their children. Preclinical models confirm this risk by showing that exposure of the developing brain to nicotine produces adverse behavioral outcomes. Here we describe behavioral phenotypes resulting from perinatal nicotine exposure in a mouse model, and discuss our findings in the context of findings from previously published studies using preclinical models of developmental nicotine exposure. METHODOLOGY/PRINCIPAL FINDINGS: Female C57Bl/6 mice received drinking water containing nicotine (100µg/ml) + saccharin (2%) starting 3 weeks prior to breeding and continuing throughout pregnancy, and until 3 weeks postpartum. Over the same period, female mice in two control groups received drinking water containing saccharin (2%) or plain drinking water. Offspring from each group were weaned at 3-weeks of age and subjected to behavioral analyses at 3 months of age. We examined spontaneous locomotor activity, anxiety-like behavior, spatial working memory, object based attention, recognition memory and impulsive-like behavior. We found significant deficits in attention and working memory only in male mice, and no significant changes in the other behavioral phenotypes in male or female mice. Exposure to saccharin alone did not produce significant changes in either sex. CONCLUSION/SIGNIFICANCE: The perinatal nicotine exposure produced significant deficits in attention and working memory in a sex-dependent manner in that the male but not female offspring displayed these behaviors. These behavioral phenotypes are associated with attention deficit hyperactivity disorder (ADHD) and have been reported in other studies that used pre- or perinatal nicotine exposure. Therefore, we suggest that preclinical models of developmental nicotine exposure could be useful tools for modeling ADHD and related disorders.

Adult Outcome of ADHD: An Overview of Results From the MGH Longitudinal Family Studies of Pediatrically and Psychiatrically Referred Youth With and Without ADHD of Both Sexes.

OBJECTIVE: We aimed to provide an overview of the Massachusetts General Hospital (MGH) Longitudinal Studies of ADHD. METHODS: We evaluated and followed samples of boys and girls with and without ADHD ascertained from psychiatric and pediatric sources and their families. RESULTS: These studies documented that ADHD in both sexes is associated with high levels of persistence into adulthood, high levels of familiality with ADHD and other psychiatric disorders, a wide range of comorbid psychiatric and cognitive disorders including mood, anxiety, and substance use disorders, learning disabilities, executive function deficits, emotional dysregulation, and autistic traits as well as functional impairments. The MGH studies suggested that stimulant treatment decreased risks of developing comorbid psychiatric disorders, substance use disorders, and functional outcomes. The MGH studies documented the neural basis of persistence of ADHD using neuroimaging. CONCLUSION: The MGH studies provided various insights on symptoms, course, functions, comorbidities, and neuroscience of ADHD.

A prenatal nicotine exposure mouse model of methylphenidate responsive ADHD-associated cognitive phenotypes.

Prenatal exposure to nicotine via cigarette smoke or other forms of tobacco use is a significant environmental risk factor for attention deficit hyperactivity disorder (ADHD). The neurobiological mechanisms underlying the link between prenatal nicotine exposure (PNE) and ADHD are not well understood. Animal models, especially rodent models, are beginning to bridge this gap in knowledge. Although ADHD is characterized by hyperactivity, inattention, impulsivity and working memory deficits, the majority of the animal models are based on only one or two ADHD associated phenotypes, in particular, hyperactivity or inattention. We report a PNE mouse model that displays the full range of ADHD associated behavioral phenotypes including working memory deficit, attention deficit and impulsive-like behavior. All of the ADHD-associated phenotypes respond to a single administration of a therapeutic equivalent dose of methylphenidate. In an earlier study, we showed that PNE produces hyperactivity, frontal cortical hypodopaminergic state and thinning of the cingulate cortex. Collectively, these data suggest that the PNE mouse model recapitulates key features of ADHD and may be a suitable preclinical model for ADHD research.

Is Maternal Smoking During Pregnancy a Risk Factor for Cigarette Smoking in Offspring? A Longitudinal Controlled Study of ADHD Children Grown Up.

OBJECTIVE: This study examined whether exposure to maternal smoking during pregnancy in children with and without ADHD is associated with smoking in offspring and whether this association is selective to ADHD children. METHOD: Ninety-six exposed and 400 unexposed participants were derived from two longitudinal studies of boys and girls with and without ADHD. Maternal smoking during pregnancy was defined by interviews with participants' mothers. RESULTS: A significant association was observed between exposure to maternal smoking in pregnancy and cigarette smoking in offspring ( p = .02). Exposed offspring were also more likely to have higher rates of major depression ( p = .04), bipolar disorder ( p = .04), and conduct disorder ( p = .04), and lower IQ ( p = .01), lower Global Assessment of Functioning (GAF) score ( p = .02), and more impaired Social Adjustment Inventory for Children and Adolescents (SAICA) scores versus unexposed offspring, adjusting for social class. CONCLUSION: Maternal smoking during pregnancy was found to increase the risk for smoking and a wide range of adverse psychiatric, cognitive, and functional outcomes in youth.

Transgenerational transmission of hyperactivity in a mouse model of ADHD.

Attention deficit hyperactivity disorder (ADHD) is a neurobehavioral disorder affecting children and adults. Genetic and environmental factors are associated with the etiology of ADHD. Among the environmental factors, exposure of the developing brain to nicotine is considered a major risk factor. Recent evidence suggests that environmental influences on the brain and behavior may be transmitted from one generation to the next. We used a prenatal nicotine exposure (PNE) mouse model of ADHD to test the hypothesis that PNE-induced hyperactivity, a proxy for human ADHD phenotype, is transmitted from one generation to the next. Our data reveal transgenerational transmission of PNE-induced hyperactivity in mice via the maternal but not the paternal line of descent. We suggest that transgenerational transmission is a plausible mechanism for propagation of environmentally induced ADHD phenotypes in the population.

Is ADHD a risk for posttraumatic stress disorder (PTSD)? Results from a large longitudinal study of referred children with and without ADHD.

OBJECTIVES: Preclinical studies link prenatal nicotine exposure with the development of both ADHD-like phenotype in rodents and blockade of extinction learning in a fear conditioning paradigm, a preclinical model of posttraumatic stress disorder (PTSD). While these findings suggest that either ADHD, prenatal nicotine exposure, or both could be a risk factor for PTSD, such associations have not been investigated in humans. METHODS: Subjects were ascertained from family-genetic, longitudinal studies of paediatrically and psychiatrically referred children with and without ADHD of both sexes and their siblings followed for 10 years from childhood into adulthood (n = 403 probands; n = 464 siblings; mean age at follow-up of probands and siblings = 22.0 years). All subjects were comprehensively evaluated with structured diagnostic interviews that included questions regarding prenatal use of cigarettes. RESULTS: A total of 12% (104/867) of the sample had been exposed to maternal smoking during pregnancy. There was no interaction effect between maternal smoking during pregnancy and ADHD (z = 0.01, P = 0.99). Maternal smoking during pregnancy and ADHD were independent, significant risk factors for PTSD at the 10-year follow-up (odds ratio = 3.58 [1.35,9.48], z = 2.57, P = 0.01 and odds ratio = 2.23 [1.06,4.69], z = 2.11, P = 0.04, respectively). CONCLUSIONS: These results suggest that both maternal smoking during pregnancy and ADHD are significant predictors of PTSD in humans.

Functional genomics of attention-deficit/hyperactivity disorder (ADHD) risk alleles on dopamine transporter binding in ADHD and healthy control subjects.

BACKGROUND: The main aim of this study was to examine the relationship between dopamine transporter (DAT) binding in the striatum in individuals with and without attention-deficit/hyperactivity disorder (ADHD), attending to the 3'-untranslated region of the gene (3'-UTR) and intron8 variable number of tandem repeats (VNTR) polymorphisms of the DAT (SLC6A3) gene. METHODS: Subjects consisted of 68 psychotropic (including stimulant)-naïve and smoking-naïve volunteers between 18 and 55 years of age (ADHD n = 34; control subjects n = 34). Striatal DAT binding was measured with positron emission tomography with 11C altropane. Genotyping of the two DAT (SLC6A3) 3'-UTR and intron8 VNTRs used standard protocols. RESULTS: The gene frequencies of each of the gene polymorphisms assessed did not differ between the ADHD and control groups. The ADHD status (t = 2.99; p<.004) and 3'-UTR of SLC6A3 9 repeat carrier status (t = 2.74; p<.008) were independently and additively associated with increased DAT binding in the caudate. The ADHD status was associated with increased striatal (caudate) DAT binding regardless of 3'-UTR genotype, and 3'-UTR genotype was associated with increased striatal (caudate) DAT binding regardless of ADHD status. In contrast, there were no significant associations between polymorphisms of DAT intron8 or the 3'-UTR-intron8 haplotype with DAT binding. CONCLUSIONS: The 3'-UTR but not intron8 VNTR genotypes were associated with increased DAT binding in both ADHD patients and healthy control subjects. Both ADHD status and the 3'-UTR polymorphism status had an additive effect on DAT binding. Our findings suggest that an ADHD risk polymorphism (3'-UTR) of SLC6A3 has functional consequences on central nervous system DAT binding in humans.

Prenatal nicotine exposure mouse model showing hyperactivity, reduced cingulate cortex volume, reduced dopamine turnover, and responsiveness to oral methylphenidate treatment.

Cigarette smoking, nicotine replacement therapy, and smokeless tobacco use during pregnancy are associated with cognitive disabilities later in life in children exposed prenatally to nicotine. The disabilities include attention deficit hyperactivity disorder (ADHD) and conduct disorder. However, the structural and neurochemical bases of these cognitive deficits remain unclear. Using a mouse model we show that prenatal nicotine exposure produces hyperactivity, selective decreases in cingulate cortical volume, and radial thickness, as well as decreased dopamine turnover in the frontal cortex. The hyperactivity occurs in both male and female offspring and peaks during the "active" or dark phase of the light/dark cycle. These features of the mouse model closely parallel the human ADHD phenotype, whether or not the ADHD is associated with prenatal nicotine exposure. A single oral, but not intraperitoneal, administration of a therapeutic equivalent dose (0.75 mg/kg) of methylphenidate decreases the hyperactivity and increases the dopamine turnover in the frontal cortex of the prenatally nicotine exposed mice, once again paralleling the therapeutic effects of this compound in ADHD subjects. Collectively, our data suggest that the prenatal nicotine exposure mouse model has striking parallels to the ADHD phenotype not only in behavioral, neuroanatomical, and neurochemical features, but also with respect to responsiveness of the behavioral phenotype to methylphenidate treatment. The behavioral, neurochemical, and anatomical biomarkers in the mouse model could be valuable for evaluating new therapies for ADHD and mechanistic investigations into its etiology.

A randomized, placebo-controlled clinical trial of bupropion for the prevention of smoking in children and adolescents with attention-deficit/hyperactivity disorder.

OBJECTIVE: Since attention-deficit/hyperactivity disorder (ADHD) is a well-documented risk factor for smoking and bupropion has been shown to be effective for smoking cessation, we tested the efficacy of bupropion as a prophylactic agent for the prevention of smoking in children and adolescents with ADHD. METHOD: We conducted a longitudinal, randomized, double-blind, placebo-controlled, parallel-group study of bupropion at a large, urban, outpatient medical center. Recruitment began in April 1999, and the last subject was followed until September 2004. Patients were nonsmoking youth, of both sexes, between 9 and 18 years of age, with DSM-IV ADHD. After random assignment to either bupropion or placebo, subjects were assessed weekly for 8 weeks, biweekly for 4 weeks, and monthly thereafter for up to 6.5 years (mean 12 months). Also, patients received treatment with psychostimulants for ADHD symptoms as needed. To assess smoking, we used an assay of cotinine in urine. RESULTS: Fifty-seven subjects (28 receiving bupropion and 29 receiving placebo) were randomly assigned and included in the analysis. No differences were found between the bupropion and placebo groups on demographic factors. About half of each group was treated with stimulants for ADHD. Statistical separation between bupropion and placebo in the rate of smoking initiation or continued smoking was not demonstrated. However, secondary post hoc analyses revealed that concurrent stimulant treatment was significantly associated with a lower rate of smoking onset (hazard ratio [HR] = 0.2, 95% CI = 0.08 to 0.89; z = -2.2, p = .03) and a lower rate of continued smoking (HR = 0.3, 95% CI = 0.11 to 0.85; z = -2.3, p = .02). CONCLUSION: While bupropion was not associated with a lower rate of smoking in youth with ADHD, post hoc analyses suggest that stimulant treatment was. Future controlled studies should investigate the role of stimulants in the prevention of smoking in children and adolescents with ADHD.

Blood pressure changes associated with medication treatment of adults with attention-deficit/hyperactivity disorder.

OBJECTIVE: To evaluate the effects of medications used in the treatment of adults with attention-deficit/hyperactivity disorder (ADHD) on blood pressure and pulse. METHOD: Subjects were those with DSM-III-R-/DSM-IV-diagnosed ADHD enrolled in placebo-controlled studies of 5 different medications for ADHD. Cardiovascular data from these studies of both stimulants (methylphenidate, amphetamine compounds, pemoline) and nonstimulants (bupropion, desipramine) were reanalyzed for baseline-to-endpoint active-treatment or placebo effects on blood pressure and heart rate. RESULTS: There were 125 subjects with a mean +/- SD age of 39 +/- 9 years. In general, active drug treatment for ADHD compared to baseline was associated with several statistically significant changes in systolic blood pressure (bupropion: +5.9 mm Hg, p < .05 by paired t test; amphetamine: +5.4 mm Hg, p < .05), diastolic blood pressure (desipramine: +7.1 mm Hg, p < .05), and heart rate (bupropion: +6.9 mm Hg, p < .05; amphetamine: +7.3 mm Hg, p < .05; methylphenidate: +4.5 mm Hg, p < .05). New-onset cases of systolic or diastolic hypertension (blood pressure > or = 140/90) were recorded in 8% (7/89) of placebo-treated subjects and 10% (9/89) of subjects receiving active medication, regardless of the class (stimulant, nonstimulant). CONCLUSION: Both stimulant and nonstimulant catecholaminergic medications used in adults with ADHD are associated with minor, but statistically significant, changes in heart rate and blood pressure that were often observed in those receiving placebo. Given the minor pressor and chronotropic effect of these medications, adults with ADHD should have their blood pressure and heart rate checked at baseline and periodically during treatment.

ADHD treatment across the life cycle.

Since attention-deficit/hyperactivity disorder (ADHD) is usually diagnosed in children, evidence from the studies of pharmacologic treatments for children with ADHD is used to inform pharmacologic treatment recommendations for adults. A large percentage of children diagnosed with ADHD have symptoms that persist into adolescence and adulthood. Evidence shows that pharmacologic treatments improve functional outcomes in children with ADHD, and studies using similar pharmacologic treatments show positive results in adults with ADHD. This article reviews the use of long-acting methylphenidate, mixed amphetamine salts, desipramine, monoamine oxidase inhibitors, bupropion, and atomoxetine in studies of children, adolescents, and adults with ADHD.

Novel treatments for attention-deficit/hyperactivity disorder in children.

Optimal medications for children with attention-deficit/hyperactivity disorder (ADHD) would be effective, well tolerated, and long acting and not cause mood swings or worsen comorbid conditions. Current medications work on brain dopamine and/or norepinephrine systems, which are thought to be involved in ADHD. The medication class with the most evidence of efficacy in ADHD is stimulants, but they may be abused, are effective for only 4 to 12 hours, and may cause mood swings or increase tic severity. In recent years, alternative treatments have been explored. Tricyclic antidepressants have efficacy comparable to that of stimulants but may cause constipation, dry mouth, tremors, blood pressure changes, and potentially serious side effects including cardiac conduction and repolarization delays. Monoamine oxidase inhibitors may improve ADHD symptoms but are associated with severe dietary restrictions. Serotonin reuptake inhibitors have little or no effect in ADHD but may improve comorbid depression. Bupropion, although less effective than stimulants, may improve both ADHD symptoms and comorbid depression. Antihypertensive agents may improve impulsivity, hyperactivity, and comorbid tics but cause sedation or rebound hypertension. Atomoxetine, which is being developed for ADHD, reduces symptoms of ADHD without exacerbating comorbid conditions and is associated with only minor side effects, including subtle changes in blood pressure and heart rate. Before prescribing a treatment, physicians should consider the appropriateness and effectiveness of any medication for children with ADHD, who may be less tolerant of side effects and less able to monitor and express concerns about their well-being than adults.