Kelly J. Huffman

Prenatal Ethanol Exposure Disrupts Intraneocortical Circuitry, Cortical Gene Expression, and Behavior in a Mouse Model of FASD

In utero ethanol exposure from a mothers consumption of alcoholic beverages impacts brain and cognitive development, creating a range of deficits in the child (Levitt, 1998; Lebel et al., 2012). Children diagnosed with fetal alcohol spectrum disorders (FASD) are often born with facial dysmorphology and may exhibit cognitive, behavioral, and motor deficits from ethanol-related neurobiological damage in early development. Prenatal ethanol exposure (PrEE) is the number one cause of preventable mental and intellectual dysfunction globally, therefore the neurobiological underpinnings warrant systematic research. We document novel anatomical and gene expression abnormalities in the neocortex of newborn mice exposed to ethanol in utero. This is the first study to demonstrate large-scale changes in intraneocortical connections and disruption of normal patterns of neocortical gene expression in any prenatal ethanol exposure animal model. Neuroanatomical defects and abnormal neocortical RZRβ, Id2, and Cadherin8 expression patterns are observed in PrEE newborns, and abnormal behavior is present in 20-d-old PrEE mice. The vast network of neocortical connections is responsible for high-level sensory and motor processing as well as complex cognitive thought and behavior in humans. Disruptions to this network from PrEE-related changes in gene expression may underlie some of the cognitive-behavioral phenotypes observed in children with FASD.

A Lifespan Analysis of Intraneocortical Connections and Gene Expression in the Mouse II

The mammalian neocortex contains an intricate processing network of multiple sensory and motor areas that allows the animal to engage in complex behaviors. These anatomically and functionally unique areas and their distinct connections arise during early development, through a process termed arealization. Both intrinsic, activity-independent and extrinsic, activity-dependent mechanisms drive arealization, much of which occurs during the areal patterning period (APP) from late embryogenesis to early postnatal life. How areal boundaries and their connections develop and change from infancy to adulthood is not known. Additionally, the adult patterns of sensory and motor ipsilateral intraneocortical connections (INCs) have not been thoroughly characterized in the mouse. In this report and its companion (I), we present the first lifespan analysis of ipsilateral INCs among multiple sensory and motor regions in mouse. We describe the neocortical expression patterns of several developmentally regulated genes that are of central importance to studies investigating the molecular regulation of arealization, from postnatal day (P) 6 to P50. In this study, we correlate the boundaries of gene expression patterns with developing areal boundaries across a lifespan, in order to better understand the nature of gene-areal relationships from early postnatal life to adulthood.

The Impact of Prenatal Ethanol Exposure on Neuroanatomical and Behavioral Development in Mice

BACKGROUND In utero alcohol, or ethanol (EtOH), exposure produces developmental abnormalities in the brain of the fetus, which can result in lifelong behavioral abnormalities. Fetal alcohol spectrum disorders (FASD) is a term used to describe a range of adverse developmental conditions caused by EtOH exposure during gestation. Children diagnosed with FASD potentially exhibit a host of phenotypes including growth retardation, facial dysmorphology, central nervous system anomalies, abnormal behavior, and cognitive deficits. Previous research suggests that abnormal gene expression and circuitry in the neocortex may underlie reported disabilities of learning, memory, and behavior resulting from early exposure to alcohol (J Neurosci, 33, 2013, 18893). METHODS Here, we utilize a mouse model of FASD to examine effects of prenatal EtOH exposure (PrEE), on brain anatomy in newborn (postnatal day [P]0), weanling (P20), and early adult (P50) mice. We correlate abnormal cortical and subcortical anatomy with atypical behavior in adult P50 PrEE mice. In this model, experimental dams self-administered a 25% EtOH solution throughout gestation (gestational days 0 to 19, day of birth), generating the exposure to the offspring. RESULTS Results from these experiments reveal long-term alterations to cortical anatomy, including atypical developmental cortical thinning, and abnormal subcortical development as a result of in utero EtOH exposure. Furthermore, offspring exposed to EtOH during the prenatal period performed poorly on behavioral tasks measuring sensorimotor integration and anxiety. CONCLUSIONS Insight from this study will help provide new information on developmental trajectories of PrEE and the biological etiologies of abnormal behavior in people diagnosed with FASD.

The developing, aging neocortex: how genetics and epigenetics influence early developmental patterning and age-related change

A hallmark of mammalian development is the generation of functional subdivisions within the nervous system. In humans, this regionalization creates a complex system that regulates behavior, cognition, memory, and emotion. During development, specification of neocortical tissue that leads to functional sensory and motor regions results from an interplay between cortically intrinsic, molecular processes, such as gene expression, and extrinsic processes regulated by sensory input. Cortical specification in mice occurs pre- and perinatally, when gene expression is robust and various anatomical distinctions are observed alongside an emergence of physiological function. After patterning, gene expression continues to shift and axonal connections mature into an adult form. The function of adult cortical gene expression may be to maintain neocortical subdivisions that were established during early patterning. As some changes in neocortical gene expression have been observed past early development into late adulthood, gene expression may also play a role in the altered neocortical function observed in age-related cognitive decline and brain dysfunction. This review provides a discussion of how neocortical gene expression and specific patterns of neocortical sensori-motor axonal connections develop and change throughout the lifespan of the animal. We posit that a role of neocortical gene expression in neocortex is to regulate plasticity mechanisms that impact critical periods for sensory and motor plasticity in aging. We describe results from several studies in aging brain that detail changes in gene expression that may relate to microstructural changes observed in brain anatomy. We discuss the role of altered glucocorticoid signaling in age-related cognitive and functional decline, as well as how aging in the brain may result from immune system activation. We describe how caloric restriction or reduction of oxidative stress may ameliorate effects of aging on the brain.

Postnatal effects of prenatal nicotine exposure on body weight, brain size and cortical connectivity in mice.

Maternal smoking results in myriad physical, cognitive, and behavioral effects in offspring due to prenatal exposure to nicotine. As the mammalian neocortex coordinates sensory integration and higher-order processes including cognition and behavioral regulation, it follows that cognitive and behavioral phenotypes of prenatal nicotine exposure (PNE) may correlate with, or stem from changes in anatomy and physiology of the neocortex. The current study uses a prenatal nicotine mouse model to determine effects of PNE on body weight, brain weight, brain length and development of neocortical circuitry, including thalamocortical afferents (TCAs) and intraneocortical connections (INCs). Although dam nutrition, dam weight gain and litter size were not significantly affected by nicotine treatment, PNE resulted in lower newborn birth weight, brain weight and length. Interestingly, the reduction of body weight, brain weight, and brain length observed in newborn PNE mice compared to control mice was no longer present at postnatal day (P) 10. A morphological study of somatosensory and visual TCAs and INCs shows no major defects in areal patterning of these connections. These data add to a growing body of literature on the neurobiological effects of PNE by providing new information on the time course of PNE-related change in the postnatal brain.

Bilateral enucleation alters gene expression and intraneocortical connections in the mouse

BackgroundAnatomically and functionally distinct sensory and motor neocortical areas form during mammalian development through a process called arealization. This process is believed to be reliant on both activity-dependent and activity-independent mechanisms. Although both mechanisms are thought to function concurrently during arealization, the nature of their interaction is not understood. To examine the potential interplay of extrinsic activity-dependent mechanisms, such as sensory input, and intrinsic activity-independent mechanisms, including gene expression in mouse neocortical development, we performed bilateral enucleations in newborn mice and conducted anatomical and molecular analyses 10 days later. In this study, by surgically removing the eyes of the newborn mouse, we examined whether early enucleation would impact normal gene expression and the development of basic anatomical features such as intraneocortical connections and cortical area boundaries in the first 10 days of life, before natural eye opening. We examined the acute effects of bilateral enucleation on the lateral geniculate nucleus of the thalamus and the neocortical somatosensory-visual area boundary through detailed analyses of intraneocortical connections and gene expression of six developmentally regulated genes at postnatal day 10.ResultsOur results demonstrate short-term plasticity on postnatal day 10 resulting from the removal of the eyes at birth, with changes in nuclear size and gene expression within the lateral geniculate nucleus as well as a shift in intraneocortical connections and ephrin A5 expression at the somatosensory-visual boundary. In this report, we highlight the correlation between positional shifts in ephrin A5 expression and improper refinement of intraneocortical connections observed at the somatosensory-visual boundary in enucleates on postnatal day 10.ConclusionsBilateral enucleation induces a positional shift of both ephrin A5 expression and intraneocortical projections at the somatosensory-visual border in only 10 days. These changes occur prior to natural eye opening, suggesting a possible role of spontaneous retinal activity in area border formation within the neocortex. Through these analyses, we gain a deeper understanding of how extrinsic activity-dependent mechanisms, particularly input from sensory organs, are integrated with intrinsic activity-independent mechanisms to regulate neocortical arealization and plasticity.

Prenatal nicotine exposure increases anxiety and modifies sensorimotor integration behaviors in adult female mice

Prenatal exposure to nicotine (PNE) has been associated with a myriad of physiological, cognitive, and behavioral effects in the developing offspring. In this study, CD-1 dams were given injections of nicotine or control vehicle throughout gestation and their offspring were raised to 6 months of age. Adult mice were administered a battery of behavioral tests (the Suok test, the elevated platform test, and the elevated plus maze test) to assess anxiety and sensorimotor integration. PNE resulted in a decreased likelihood of jumping during the elevated platform test and decreased directed exploration in the Suok test, both indicative of increased anxiety. Also, PNE mice showed increased numbers of missteps while traversing an elevated rod in the Suok test, demonstrating altered sensorimotor integration. No significant differences were found in falls, segments traveled, latency to leave the central zone, vegetative responses, risk assessment behaviors, or autogroom behaviors. The elevated plus maze test revealed no significant differences between groups. No significant differences in body and brain weights, or cortical thickness within motor, somatosensory, and visual cortices were observed between PNE and control mice. Although neuroanatomical effects of PNE may be rescued as development progresses, defects in sensorimotor integration and increased anxiety persist into adulthood.

Prenatal Ethanol Exposure and Neocortical Development: A Transgenerational Model of FASD

Fetal Alcohol Spectrum Disorders, or FASD, represent a range of adverse developmental conditions caused by prenatal ethanol exposure (PrEE) from maternal consumption of alcohol. PrEE induces neurobiological damage in the developing brain leading to cognitive-perceptual and behavioral deficits in the offspring. Alcohol-mediated alterations to epigenetic function may underlie PrEE-related brain dysfunction, with these changes potentially carried across generations to unexposed offspring. To determine the transgenerational impact of PrEE on neocortical development, we generated a mouse model of FASD and identified numerous stable phenotypes transmitted via the male germline to the unexposed third generation. These include alterations in ectopic intraneocortical connectivity, upregulation of neocortical Rzrβ and Id2 expression accompanied by both promoter hypomethylation of these genes and decreased global DNA methylation levels. DNMT expression was also suppressed in newborn PrEE cortex, providing further insight into how ethanol perturbs DNA methylation leading to altered regulation of gene transcription. These PrEE-induced, transgenerational phenotypes may be responsible for cognitive, sensorimotor, and behavioral deficits seen in humans with FASD. Thus, understanding the possible epigenetic mechanisms by which these phenotypes are generated may reveal novel targets for therapeutic intervention of FASD and lead to advances in human health.

The effects of lifelong blindness on murine neuroanatomy and gene expression.

Mammalian neocortical development is regulated by neural patterning mechanisms, with distinct sensory and motor areas arising through the process of arealization. This development occurs alongside developing central or peripheral sensory systems. Specifically, the parcellation of neocortex into specific areas of distinct cytoarchitecture, connectivity and function during development is reliant upon both cortically intrinsic mechanisms, such as gene expression, and extrinsic processes, such as input from the sensory receptors. This developmental program shifts from patterning to maintenance as the animal ages and is believed to be active throughout life, where the brain’s organization is stable yet plastic. In this study, we characterize the long-term effects of early removal of visual input via bilateral enucleation at birth. To understand the long-term effects of early blindness we conducted anatomical and molecular assays 18 months after enucleation, near the end of lifespan in the mouse. Bilateral enucleation early in life leads to long-term, stable size reductions of the thalamic lateral geniculate nucleus (LGN) and the primary visual cortex (V1) alongside a increase in individual whisker barrel size. Neocortical gene expression in the aging brain has not been previously identified; we document cortical expression of multiple regionalization genes. Expression patterns of Ephrin A5, COUP-TFI, and RZRβ and patterns of intraneocortical connectivity (INC) are altered in the neocortices of aging blind mice. Sensory inputs from different modalities during development likely play a major role in the development of cortical areal and thalamic nuclear boundaries. We suggest that early patterning by prenatal retinal activity combined with persistent gene expression within the thalamus and cortex is sufficient to establish and preserve a small but present LGN and V1 into late adulthood.

Rapid Changes in Cortical and Subcortical Brain Regions after Early Bilateral Enucleation in the Mouse.

Functional sensory and motor areas in the developing mammalian neocortex are formed through a complex interaction of cortically intrinsic mechanisms, such as gene expression, and cortically extrinsic mechanisms such as those mediated by thalamic input from the senses. Both intrinsic and extrinsic mechanisms are believed to be involved in cortical patterning and the establishment of areal boundaries in early development; however, the nature of the interaction between intrinsic and extrinsic processes is not well understood. In a previous study, we used a perinatal bilateral enucleation mouse model to test some aspects of this interaction by reweighting sensory input to the developing cortex. Visual deprivation at birth resulted in a shift of intraneocortical connections (INCs) that aligned with ectopic ephrin A5 expression in the same location ten days later at postnatal day (P) 10. A prevailing question remained: Does visual deprivation first induce a change in gene expression, followed by a shift in INCs, or vice versa? In the present study, we address this question by investigating the neuroanatomy and patterns of gene expression in post-natal day (P) 1 and 4 mice following bilateral enucleation at birth. Our results demonstrate a rapid reduction in dorsal lateral geniculate nucleus (dLGN) size and ephrin A5 gene expression 24-hours post-enucleation, with more profound effects apparent at P4. The reduced nuclear size and diminished gene expression mirrors subtle changes in ephrin A5 expression evident in P1 and P4 enucleated neocortex, 11 and 8 days prior to natural eye opening, respectively. Somatosensory and visual INCs were indistinguishable between P1 and P4 mice bilaterally enucleated at birth, indicating that perinatal bilateral enucleation initiates a rapid change in gene expression (within one day) followed by an alteration of sensory INCs later on (second postnatal week). With these results, we gain a deeper understanding of how gene expression and sensory input together regulate cortical arealization and plasticity during early development.