NMARC Current Research and Core Components

Principal Investigator: Erin Milligan, Ph.D.
Co-Investigators: Andrea Allan, Ph.D., Kevin Caldwell, Ph.D. & Nikolaos Mellios, M.D., Ph.D.

Many of the physiologic processes affected by PAE are regulated by glucocorticoids (GCs).  GC resistance (i.e., reduced sensitivity to the actions of GCs) is associated with a variety of chronic diseases, several of which are also associated with PAE (e.g., immune and inflammatory disorders, insulin resistance).  Life-long GC responsiveness is established in utero as the result of aberrant glucocorticoid programming.  We hypothesize that PAE influences GR programming by increasing fetal brain Growth arrest-specific 5 (Gas5), an abundantly expressed long non-coding RNA that binds to the glucocorticoid receptor (GR) DNA-binding domain, thus inhibiting GR-dependent transcriptional regulation.  Because Gas5 levels are greatly reduced postnatally, we propose that FK506-binding protein 51 (Fkbp51) replaces Gas5 as the primary regulator of GR-dependent signaling in the adult and is responsible for continued GC resistance.  We will test the hypotheses using our established mouse model of PAE.  In Aim 1 we will assess the effects of PAE on several measures of glucocorticoid resistance, including effects on dexamethasone suppression of corticosterone responses, pro- and anti-inflammatory cytokine levels, levels of Fkbp5 and Gas5 promoter methylation, and levels of various specific GR-regulated gene transcripts.  In Aim 2 we will determine the effects of PAE on developmental programming of GC responding during the stress and the immune hyporesponsive periods, which occur during the preweanling period in mouse.  In Aim 3 we will use targeted in utero interventions to test the hypothesis that inhibition or reduction of prenatal Gas-5 will restore normal GC sensitivity in the PAE mice.

Co-Principal Investigators: Daniel Savage, Ph.D. & Derek Hamilton
Co-Investigator: Fernando Valenzuela, Ph.D.

We have observed that the histamine H₃ receptor inverse agonist ABT-239 ameliorates PAE-induced deficits in dentate gyrus (DG) long-term potentiation (LTP) and retention of memory in rats.  Further, PAE also increases H₃ receptor-effector coupling and heightens H₃ receptor-mediated inhibition of glutamate release in the DG (Varaschin et al., 2018).  We hypothesize that a PAE-induced differential expression of H₃ receptor isoforms is the basis for heightened H₃ receptor-effector coupling and that treatment with a H₃ receptor inverse agonist, SAR152954, will blunt these PAE-induced heightened inhibitory responses and ameliorate PAE-induced deficits in glutamate release and LTP.  In Aim 1, we will examine the effects of PAE on the mRNA and protein expression of the rH3A and rH3C isoforms of H₃ receptors and examine how PAE alters responsiveness to the H₃ agonist methimepip and to SAR152954.  In Aim 2A, we will examine how PAE alters dentate granule cell responsiveness to methimepip and SAR152954 in vitro.  In Aim 2B, we will extend these studies to recordings of physiological responses combined with changes in glutamate levels in dentate gyrus of awake freely moving rats.

Principal Investigator: Jonathan Brigman, Ph.D.
Co-Investigator: Fernando Valenzuela, Ph.D

Impairments in executive control have come into increasing focus in FASD due to the strong negative impact on quality of life measures.  Working closely with Dr. Julia Stephen (Component 5), we are increasingly focusing on the executive control domains of attention and cognitive control.  Clinically, cognitive control is measured via continuous performance tasks (CPT) which present multiple stimulus types and require subjects to respond to targets, but inhibit responding to non-target stimuli.  We have recently validated a rodent touch-screen five choice CPT (5C-CPT) and have evidence for strong homology of parietal beta oscillatory signaling during performance from electroencephalogram (EEG) in healthy human subjects and EEG-like dura recording in mice.  While new clinical data from Dr. Stephen suggest that PAE impairs the ability to withhold responding on CPT, the mechanisms underlying these deficits are not understood.  Therefore, Component 4 will test whether attention and response inhibition utilizing a touch-screen 5C-CPT are impaired in the mouse model of PAE.  Given strong preliminary evidence that PAE does impair these behaviors, we propose to perform EEG-like recording at the dura and recording within the cortex to examine whether PAE significantly alters beta oscillatory signaling and if alterations are related to effects on neuronal firing pattern or timing.  There is strong evidence that high-frequency oscillations are controlled by specific populations of cortical interneurons and numerous studies have shown these populations are altered by PAE.  Therefore, we will examine alterations in interneurons as a putative mechanism of deficits on CPT tasks by examining alterations in number and function of these neuronal cell types.

Co-Principal Investigators: Julia Stephen Ph.D. & Dina Hill Ph.D.

We will capitalize on the strengths of the broader P50 team to examine the underlying mechanisms that lead to cognitive and behavioral deficits in children with FASD, with an emphasis on inhibitory function.  We previously employed the SART to differentiate children with ADHD from children with FASD, thereby examining differences in brain function based on attention deficits revealed in both populations.  Interestingly, this task can also be used to examine inhibitory function as a Go/No-Go task.  Our goal in the Component 5 Project is to examine inhibitory function in young children with FASD relative to healthy controls to understand the role of neural oscillations with the goal of identifying treatment targets for children with FASD.  In combination with examining task-evoked neural oscillations and task-evoked connectivity measures, we will also examine resting state connectivity using both fMRI and MEG.  Our premise is that neural oscillations play a primary role in mediating one’s ability to respond appropriately to stimuli during resting state conditions.  In combination with two of the three preclinical projects, we will examine the role of neural oscillations in PAE.  In contrast to our prior study, we will now examine children in the 6-8 year age range.  We are particularly motivated to study children in this age range based on the developmental trajectory of inhibitory function in children.  The ability to inhibit responses according to task instructions develops by four years of age and the ability to successfully inhibit responses continues to develop through adolescence.  Yet, this executive function is highly correlated with success in both the academic and social domains.  Therefore, we posit that identifying markers of poor inhibitory functioning early in the developmental trajectory will provide the best time window for successful interventions.  Understanding the mechanisms that lead to this failure in inhibitory function is a key factor in developing optimal interventions.

The Prenatal Exposures Clinic provides multidisciplinary evaluations for children who are prenatally exposed to alcohol and/or other substances of abuse to determine a diagnosis on the Fetal Alcohol Spectrum Disorder (FASD) spectrum or other disorders related to prenatal exposure. These evaluations include a brief physical exam, interview with caregivers, and a screening of the child’s cognitive skills and behavioral/adaptive functioning. Our evaluation team includes a pediatrician, neuropsychologist, occupational therapist, social worker, and a psychologist.

Our follow-up services include comprehensive neuropsychological testing and educational consultation for school-aged children and adolescents.  A neuropsychological evaluation will provide a description of a child’s individual learning and behavioral profile along with recommendations for behavioral interventions and academic teaching strategies to meet a child’s unique learning needs. We are able to attend IEP meetings and consult directly with school personnel.

Principal Investigator: Sharon L. Ruyak, Associate Professor, College of Nursing

Prenatal alcohol exposure (PAE) is associated with a continuum of disabilities known as fetal alcohol spectrum disorder (FASD). Prevalence estimates of FASD range from 1.1% to 5% among school age children making it one of the top forms of preventable developmental disabilities in the U.S. Almost 40% of women in the U.S. reported exposure to multiple adverse childhood experiences (ACE), and among those who self-report ≥ 4 ACE, the risk of prenatal alcohol consumption is almost 5 times higher. But, there is limited investigation of the synergistic influence of PAE and ACE on risk for development of FASD highlighting the critical gap in our understanding of the factors that influence susceptibility to FASD. Epigenetic mechanisms, such as microRNA (miRNA) that regulate gene expression, are strongly favored as mechanisms that mediate the transmission of long-lasting effects of both PAE and maternal ACE to the developing fetus. Moreover, miRNAs have been shown to influence placental structure and function. The placenta is a critical interface between the mother and fetus facilitating the exchange of oxygen, nutrients, hormones and waste. Studies demonstrate bidirectional traffic of maternal and fetal cells across the placenta, thus maternal circulating miRNAs may influence offspring outcomes via this unique interface. Our long-term goal is to understand the underlying mechanisms that influence susceptibility to FASD in order to inform development of targeted prevention and treatment strategies for women and infants affected by PAE. The objective of this pilot proposal is to examine maternal circulating miRNAs associated with placental health and function that differ in expression levels among individuals who consume alcohol during pregnancy with and without a significant history of ACE. We will accomplish this through: 1) characterization of differential miRNA expression profiles in maternal circulation as a consequence of PAE and ACE using miRNA sequencing and 2) identification of novel signaling pathways associated with PAE and ACE interaction through enrichment analysis of target genes to identify possible common molecular pathways involved in placental development, health, and function. Completion of the proposed work will provide preliminary data for an R01 submission to the NIH that will examine the role of placental function, and the influence of maternal circulating factors on placental function, in the variability of newborn neurobehavioral outcomes dation for early touchpoints for identification of high-risk mothers and infants, as well as possible targets for early intervention to mitigate the effects of PAE.

Principal Investigator: Nan Zeng, Research Assistant Professor, Prevention and Population Sciences, Department of Pediatrics

Children diagnosed with a FASD may demonstrate a wide range of deficits, including physical, cognitive, behavioral, and psychosocial problems that can impact their ability to function in society. Therefore, intervention programs aim to promote health in this population is warranted. One intervention strategy that may be promising for improving multiple areas of the lives of individuals with a FASD is physical activity (PA). Aside from the general health benefits of an active lifestyle (e.g., cardiovascular benefits), PA can also improve various aspects of cognitive function, notably executive function, and appears to be effective across the life span. Today’s children are living in the age of technology, where active video games (i.e., exergaming) are the ideal solution to promote and encourage more energy expenditure during their free time. Despite the potential health benefits of commercially available Exergaming in children, this PA modality has not been well studied as an intervention strategy for children with a FASD. This pilot randomized controlled trial (RCT) aims to develop, implement, and evaluate an 8-week, home-based, and family-centered RCT employing exergaming to promote movement behaviors as well as inhibitory control and attention in families of children with FASD. We plan to use LeapTV™ --- a commercially available educational Exergaming system designed for children ages three to eight years that accentuates learning through motion. We aim to recruit 16 parent-child dyads to evaluate 1) the feasibility, acceptability, appropriateness, and potential for the adoption and sustainment of Exergaming intervention from the perspective of parents, and 2) preliminary efficacy of such intervention on children’s physical and bio-psycho-social health benefits. This PA intervention program is innovative due to its unique approach of incorporating the concept of a healthy family environment into exergaming to assess its impact on compliance, sustainability, and transferability. Successful completion of this pilot study will provide the empirical basis for this exploratory design process. The findings from this pilot study will inform the appropriate use of exergaming in children with a FASD, as well as lay the groundwork for future large-scale multi-level multi-component family-based health media intervention trials, which may then be adapted or translated into school or community settings and can have a significant public health impact.

Principal Investigator: Valentina Licheri, Adjunct Assistant Professor, Department of Neurosciences

Clinical studies have reported that up to 80% of children diagnosed with Fetal Alcohol Spectrum Disorders (FASDs) exhibit significant sleep disorders including short sleep duration, sleep anxiety, bedtime resistance, increased sleep fragmentation and parasomnias which may affect development of cognitive functions and disruptive behaviours. Despite the several preclinical and clinical studies showing the effects of prenatal alcohol exposure (PAE) on sleep- wake behavior, the molecular mechanisms are not well understood. Surprisingly, relatively few studies with animal models of FASDs have characterized the effect of developmental ethanol exposure on sleep physiology and these have focused on high doses, producing blood concentrations between 167-500 mg/dl. However, the effects of moderate PAE, which commonly occurs in many populations in the USA and across the world have yet to be characterized. Furthermore, considerable progress has been made with machine learning-based methodologies to study sleep physiology, which have uncovered three novel sub-stages of REM and NREM sleep in rodents model that closely resemble human sleep stages. Our recent investigations demonstrated that low-moderate gestational alcohol exposure is able to affect cognitive behaviors and cortical function in a sex-specific manner. Moderate PAE altered glial glutamate transporters in adolescent male offspring, which are involved in the control of extracellular glutamate across sleep and waking states. Gestational alcohol exposure induces a significant decrease in the mGluR5R protein, a glutamate receptor involved in sleep regulation. Interneuronopathy has been observed in PAE models, suggesting a possible correlation with sleeping disorders considering the critical role of cortical interneurons across sleep states. I hypothesize that moderate alcohol exposure during the rodent equivalent to the first and second trimesters of human pregnancy disrupts sleep physiology in adolescent offspring. To test this hypothesis, I will study the sleep-wake cycle in adolescent PAE male and female mice (postnatal day 40-60) performing electroencephalography (EGG) and electromyography (EMG) recordings, and characterize the sleep sub-stages using AI-based methods. This 1-year pilot project will represent the first characterization of the impact of moderate PAE model on sleep architecture, and providing new experimental framework to characterize the molecular pathways involved in the sleeping disorders observed in FASD.