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.

Hippocampal neuroinflammation following combined exposure to cyclophosphamide and naproxen in ovariectomized mice.

Aim: Cancer patients undergoing chemotherapy report cognitive changes collectively termed "chemo brain." Neuroinflammation is among the factors believed to contribute to "chemo brain" suggesting a potential beneficial role for anti-inflammatory drugs in cancer patients undergoing chemotherapy. We investigated whether the non-steroidal anti-inflammatory drug naproxen influenced hippocampal inflammation in non-tumor bearing female mice receiving the chemotherapy drug cyclophosphamide (CP).Materials and methods: Intact and ovariectomized C57BL/6 mice were used to examine potential role of ovarian hormones on neuroinflammation. The mice were placed on naproxen (375 ppm) or control diet, and a week later CP (100 mg/kg; i.p.) was administered every 3 days for 2 weeks. We analyzed hippocampal inflammatory biomarkers, anxiety-like behavior, spatial working memory, exploratory behavior, spontaneous locomotor activity and depression-like behavior.Results: CP produced significant effects on anti-inflammatory but not pro-inflammatory biomarkers. However, CP and naproxen in combination produced significant effects on both pro- and anti- inflammatory biomarkers. Naproxen and ovariectomy individually produced significant effects on pro- and anti-inflammatory biomarkers as well. Working memory and depression-like behavior were not significantly influenced by CP, naproxen or ovariectomy individually although CP and ovariectomy produced significant interaction effects on depression-like behavior. Exploratory behavior and locomotor activity showed significant effects of CP, and interaction between CP and naproxen was significant for locomotor activity.Conclusions: Ovariectomy, naproxen and a combination of CP and naproxen upregulate hippocampal pro- and anti- inflammatory biomarkers. None of the factors individually produce significant behavioral changes that could be consistent with chemo brain, although CP and ovariectomy in combination produced significant effects on depression-like behavior, a co-morbidity of chemo brain.

Nicotine and the developing brain: Insights from preclinical models.

Use of tobacco products during pregnancy is associated with increased risk for neurodevelopmental disorders in the offspring. Preclinical models of developmental nicotine exposure have offered valuable insights into the neurobiology of nicotine's effects on the developing brain and demonstrated lasting effects of developmental nicotine exposure on brain structure, neurotransmitter signaling and behavior. These models have facilitated discovery of novel compounds as candidate treatments for attention deficit hyperactivity disorder, a neurodevelopmental disorder associated with prenatal nicotine exposure. Using these models the significance of heritability of behavioral phenotypes from the nicotine-exposed pregnant female or adult male to multiple generations of descendants has been demonstrated. Finally, research using the preclinical models has demonstrated synergistic interactions between developmental nicotine exposure and repetitive mild traumatic brain injury that contribute to "worse" outcomes from the injury in individuals with attention deficit hyperactivity disorder associated with developmental nicotine exposure.

Repetitive Mild Traumatic Brain Injury in an Awake, Unanesthetized Mouse Model of Perinatal Nicotine Exposure Produces Transient Novelty-Seeking and Depression-Like Behaviors.

Attention deficit hyperactivity disorder (ADHD) can be a risk factor for repetitive mild traumatic brain injury (mTBI) or concussions such as those that can occur in contact sports. Individuals with ADHD also appear to have a higher risk of poor neurocognitive outcomes after repetitive mTBI. Findings from clinical studies examining the interactions between ADHD and repetitive mTBI vary, likely because of variabilities in experimental design and outcome measures. We used a mouse model of perinatal nicotine exposure (PNE), which displays behavioral, neuroanatomical, and neurotransmitter features consistent with ADHD and subjected the mice to repetitive mTBI. We used a closed head model of mTBI in awake, unanesthetized mice to mimic concussions in humans. The mTBI was repeated three times daily for seven days. The mice in the PNE-mTBI group took longer to regain consciousness after the mTBI and showed transient novelty-seeking and depression-like behaviors. Before the repetitive mTBI, the mice in the PNE group showed attention deficit, which persisted after the mTBI. The mice in the control (non-PNE) group showed a transient attention deficit after the repetitive mTBI but not any of the other behavioral changes seen in the PNE-mTBI group. These findings from an unanesthetized mouse model with a pre-existing condition show that ADHD and repetitive mTBI together contribute to transient novelty-seeking and depression-like behavior supporting the notion that untreated ADHD may be a risk factor for poor neurocognitive outcomes after concussions.

Heritable consequences of paternal nicotine exposure: from phenomena to mechanisms†.

Our understanding of the interactions between genetic and environmental factors in shaping behavioral phenotypes has expanded to include environment-induced epigenetic modifications and the intriguing possibility of their association with heritable behavioral phenotypes. The molecular basis of heritability of phenotypes arising from environment-induced epigenetic modifications is not well defined yet. However, phenomenological evidence in favor of it is accumulating rapidly. The resurgence of interest has led to focus on epigenetic modification of germ cells as a plausible mechanism of heritability. Perhaps partly because of practical reasons such as ease of access to male germ cells compared to female germ cells, attention has turned toward heritable effects of environmental influences on male founders. Public health implications of heritable effects of paternal exposures to addictive substances or to psycho-social factors may be enormous. Considering nicotine alone, over a billion people worldwide use nicotine-containing products, and the majority are men. Historically, the adverse effects of nicotine use by pregnant women received much attention by scientists and public policy experts alike. The implications of nicotine use by men for the physical and mental well-being of their children were not at the forefront of research until recently. Here, we review progress in the emerging field of heritable effects of paternal nicotine exposure and its implications for behavioral health of individuals in multiple generations.

Repetitive Mild Traumatic Brain Injury in a Perinatal Nicotine Exposure Mouse Model of Attention Deficit Hyperactivity Disorder.

Attention deficit hyperactivity disorder (ADHD) increases the risk for concussion or mild traumatic brain injury (mTBI). At the same time, recommendations for the management of ADHD include participation in sports and other organized physical activities, including those that carry an increased risk of mTBI. Very little work has been done to determine the extent to which untreated ADHD adversely impacts behavioral outcomes of repeated mild concussions. Here, we used a perinatal nicotine exposure (PNE) mouse model of ADHD combined with a closed-head, repetitive mTBI model. The PNE mouse model carries significant construct, face, and predictive validity as a preclinical model of ADHD. Two-month-old PNE and control mice were subjected to closed-head repetitive mTBI or sham procedure once daily for 5 days. Object-based attention, novel object recognition memory, spatial working memory, and depression-like behavior were analyzed 1 day and 2 weeks following repeated mTBI. Consistent with our previous reports, mice in the PNE group showed significant deficits in object-based attention and working memory prior to mTBI. These deficits persisted following the repeated mTBI. Repeated mTBI produced a transient attention deficit in the control group but did not exacerbate the attention deficit that is characteristic of the PNE group. Although neither PNE nor repetitive mTBI alone influenced immobility in the tail suspension test, when PNE mice were subjected to mTBI, there was a transient increase in this measurement suggesting a synergistic effect of ADHD and mTBI on depression-like behavior. Thus, our data using the PNE mouse model suggest that ADHD may be a risk factor for transient depression following repeated mTBI and that repeated mTBI may be a risk factor for transient attention deficit.

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.

The adenosine A(2A) receptor agonist CGS 21680 alleviates auditory sensorimotor gating deficits and increases in accumbal CREB in rats neonatally treated with quinpirole.

RATIONALE AND OBJECTIVE: The adenosine A(2A) receptor forms a mutually inhibitory heteromer with the dopamine D2 receptor, and A(2A) agonists decrease D2 signaling. This study analyzed whether an adenosine A(2A) agonist would alleviate deficits in sensorimotor gating and increases in cyclic-AMP response element binding protein (CREB) in the nucleus accumbens (NAc) in the neonatal quinpirole model of schizophrenia (SZ). METHODS: Male and female Sprague-Dawley rats were neonatally treated with saline (NS) or quinpirole HCl (NQ; 1 mg/kg) from postnatal days (P) 1-21. Animals were raised to P44 and behaviorally tested on auditory sensorimotor gating as measured through prepulse inhibition (PPI) from P44 to P48. Approximately 15 min before each session, animals were given an ip administration of saline or the adenosine A(2A) agonist CGS 21680 (0.03 or 0.09 mg/kg). One day after PPI was complete on P49, animals were administered a locomotor activity test in the open field after saline or CGS 21680 treatment, respectively. On P50, the nucleus accumbens (NAc) was evaluated for CREB protein. RESULTS: NQ-treated rats demonstrated a deficit in PPI that was alleviated to control levels by either dose of CGS 21680. The 0.03 mg/kg dose of CGS 21680 increased startle amplitude in males. The 0.09 mg/kg dose of CGS 21680 resulted in an overall decrease in locomotor activity. NQ treatment significantly increased NAc CREB that was attenuated to control levels by either dose of CGS 21680. CONCLUSIONS: This study revealed that an adenosine A(2A) receptor agonist was effective to alleviate PPI deficits in the NQ model of SZ in both male and female rats.

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.

Nicotine in Senescence and Atherosclerosis.

Cigarette smoke is a known exacerbator of age-related pathologies, such as cardiovascular disease (CVD), atherosclerosis, and cellular aging (senescence). However, the role of nicotine and its major metabolite cotinine is yet to be elucidated. Considering the growing amount of nicotine-containing aerosol use in recent years, the role of nicotine is a relevant public health concern. A number of recent studies and health education sites have focused on nicotine aerosol-induced adverse lung function, and neglected cardiovascular (CV) impairments and diseases. A critical review of the present scientific literature leads to the hypothesis that nicotine mediates the effects of cigarette smoke in the CV system by increasing MAPK signaling, inflammation, and oxidative stress through NADPH oxidase 1 (Nox1), to induce vascular smooth muscle cell (VSMC) senescence. The accumulation of senescent VSMCs in the lesion cap is detrimental as it increases the pathogenesis of atherosclerosis by promoting an unstable plaque phenotype. Therefore, nicotine, and most likely its metabolite cotinine, adversely influence atherosclerosis.

Effects of Developmental Nicotine Exposure on Frontal Cortical GABA-to-Non-GABA Neuron Ratio and Novelty-Seeking Behavior.

Cigarette smoking during pregnancy is a major public health concern, resulting in detrimental health effects in the mother and her offspring. The adverse behavioral consequences for children include increased risk for attention deficit hyperactivity disorder, working memory deficits, epilepsy, novelty-seeking, and risk-taking behaviors. Some of these behavioral conditions are consistent with an imbalance in frontal cortical excitatory (glutamate) and inhibitory (GABA) neurotransmitter signaling. We used a GAD67-GFP knock-in mouse model to examine if developmental nicotine exposure alters frontal cortical GABA neuron numbers, GABA-to-non-GABA neuron ratio and behavioral phenotypes. Female mice were exposed to nicotine (100 or 200 µg/mL) in drinking water beginning 3 weeks prior to breeding and until 3 weeks postpartum. Male and female offspring were examined beginning at 60 days of age. The nicotine exposure produced dose-dependent decreases in GABA-to-non-GABA neuron ratios in the prefrontal and medial prefrontal cortices without perturbing the intrinsic differences in cortical thickness and laminar distribution of GABA or non-GABA neurons between these regions. A significant increase in exploratory behavior and a shift toward "approach" in the approach-avoidance paradigm were also observed. Thus, developmental nicotine exposure shifts the cortical excitation-inhibition balance toward excitation and produces behavioral changes consistent with novelty-seeking behavior.

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.

Translational effects and coding potential of an upstream open reading frame associated with DOPA Responsive Dystonia.

Upstream open reading frames (uORFs) have emerged as major post-transcriptional regulatory elements in eukaryotic species. In general, uORFs are initiated by a translation start codon within the 5' untranslated region of a gene (upstream ATG; uATG), and they are negatively correlated with translational efficiency. In addition to their translational regulatory role, some uORFs can code for biologically active short peptides. The importance of uATGs/uORFs is further underscored by human diseases associated with single nucleotide polymorphisms (SNPs), which disrupt existing uORFs or introduce novel uORFs. Although several functional proteins translated from naturally occurring uORFs have been described, the coding potential of uORFs created by SNPs has been ignored because of the a priori assumption that these proteins are short-lived with no likely impact on protein homeostasis. Thus, studies on SNP-created uORFs are limited to their translational effects, leaving unexplored the potential cellular consequences of a SNP/uORF-encoded protein. Here, we investigate functionality of a uATG/uORF introduced by a +142C>T SNP within the GCH1 gene and associated with a familial form of DOPA Responsive Dystonia. We report that the +142C>T SNP represses GCH1 translation, and introduces a short, frame shifted uORF that encodes a 73-amino acid peptide. This peptide is localized within the nucleus and compromises cell viability upon proteasome inhibition. Our work extends the list of uATG/uORF associated diseases and advances research on peptides translated from SNP-introduced uORFs, a neglected component of the proteome.

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.

Nucleosome Repositioning: A Novel Mechanism for Nicotine- and Cocaine-Induced Epigenetic Changes.

Drugs of abuse modify behavior by altering gene expression in the brain. Gene expression can be regulated by changes in DNA methylation as well as by histone modifications, which alter chromatin structure, DNA compaction and DNA accessibility. In order to better understand the molecular mechanisms directing drug-induced changes in chromatin structure, we examined DNA-nucleosome interactions within promoter regions of 858 genes in human neuroblastoma cells (SH-SY5Y) exposed to nicotine or cocaine. Widespread, drug- and time-resolved repositioning of nucleosomes was identified at the transcription start site and promoter region of multiple genes. Nicotine and cocaine produced unique and shared changes in terms of the numbers and types of genes affected, as well as repositioning of nucleosomes at sites which could increase or decrease the probability of gene expression based on DNA accessibility. Half of the drug-induced nucleosome positions approximated a theoretical model of nucleosome occupancy based on physical and chemical characteristics of the DNA sequence, whereas the basal or drug naïve positions were generally DNA sequence independent. Thus we suggest that nucleosome repositioning represents an initial dynamic genome-wide alteration of the transcriptional landscape preceding more selective downstream transcriptional reprogramming, which ultimately characterizes the cell- and tissue-specific responses to drugs of abuse.

New methods for investigation of neuronal migration in embryonic brain explants.

BACKGROUND: Proper migration of neurons is essential for the formation and normal functioning of the nervous system. Defects in neuronal migration underlie a number of neurologic diseases in humans. Although cell migration is crucial for neural development, molecular mechanisms guiding neuronal migration remain to be elucidated fully. Newborn neurons from the embryonic medial ganglionic eminence (MGE) migrate a long distance dorsally in the developing brain, giving rise to several types of interneurons in the neocortex. NEW METHOD: In this study, we developed an immunocytochemistry (ICC) protocol to stain neurons migrating out of the MGE explant embedded in Matrigel. We also established a protocol to efficiently transfect cells in MGE explants, achieving a transduction efficiency of more than 30%. COMPARISON WITH EXISTING METHOD: In addition, we developed microfluidic chambers for explants that allow visualization of the vectorial migration of individual neurons from mouse embryonic MGE explants. Our microfluidic system allows monitoring of the distribution of cellular organelles (e.g. Golgi) within migrating neurons which have been stained with commercial molecular dyes or transfected with adeno-associated virus (AAV) expressing reporter proteins. CONCLUSION: These methods provide new paradigms to study neuronal migration in real-time.

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.

Cup of Joe: a brain development "no"?

Treating pregnant mice with adenosine receptor antagonists including caffeine results in delayed migration of cortical γ-aminobutyric acid neurons and altered brain development in mouse offspring (Silva et al.).