Virtual Histology of Transgenic Mouse Embryos for High-Throughput Phenotyping

A bold new effort to disrupt every gene in the mouse genome necessitates systematic, interdisciplinary approaches to analyzing patterning defects in the mouse embryo. We present a novel, rapid, and inexpensive method for obtaining high-resolution virtual histology for phenotypic assessment of mouse embryos. Using osmium tetroxide to differentially stain tissues followed by volumetric X-ray computed tomography to image whole embryos, isometric resolutions of 27 μm or 8 μm were achieved with scan times of 2 h or 12 h, respectively, using mid-gestation E9.5–E12.5 embryos. The datasets generated by this method are immediately amenable to state-of-the-art computational methods of organ patterning analysis. This technique to assess embryo anatomy represents a significant improvement in resolution, time, and expense for the quantitative, three-dimensional analysis of developmental patterning defects attributed to genetically engineered mutations and chemically induced embryotoxicity

Use of High Resolution 3D Diffusion Tensor Imaging to Study Brain White Matter Development in Live Neonatal Rats

High resolution diffusion tensor imaging (DTI) can provide important information on brain development, yet it is challenging in live neonatal rats due to the small size of neonatal brain and motion-sensitive nature of DTI. Imaging in live neonatal rats has clear advantages over fixed brain scans, as longitudinal and functional studies would be feasible to understand neuro-developmental abnormalities. In this study, we developed imaging strategies that can be used to obtain high resolution 3D DTI images in live neonatal rats at postnatal day 5 (PND5) and PND14, using only 3 h of imaging acquisition time. An optimized 3D DTI pulse sequence and appropriate animal setup to minimize physiological motion artifacts are the keys to successful high resolution 3D DTI imaging. Thus, a 3D rapid acquisition relaxation enhancement DTI sequence with twin navigator echoes was implemented to accelerate imaging acquisition time and minimize motion artifacts. It has been suggested that neonatal mammals possess a unique ability to tolerate mild-to-moderate hypothermia and hypoxia without long term impact. Thus, we additionally utilized this ability to minimize motion artifacts in magnetic resonance images by carefully suppressing the respiratory rate to around 15/min for PND5 and 30/min for PND14 using mild-to-moderate hypothermia. These imaging strategies have been successfully implemented to study how the effect of cocaine exposure in dams might affect brain development in their rat pups. Image quality resulting from this in vivo DTI study was comparable to ex vivo scans. fractional anisotropy values were also similar between the live and fixed brain scans. The capability of acquiring high quality in vivo DTI imaging offers a valuable opportunity to study many neurological disorders in brain development in an authentic living environment

J Magn Reson Imaging

PurposeChronic hypoxemia is the prime cause of fetal brain injury and long-term sequelae such as neurodevelopmental compromise, seizures and cerebral palsy. This study aims to investigate the impact of chronic hypoxemia on neonatal brains, and follow developmental alterations and adaptations non-invasively in a guinea pig model.Materials and MethodsThirty guinea pigs underwent either normoxic and hypoxemic conditions during the critical stage of brain development (0.7 gestation) and studied prenatally (n=16) or perinatally (n=14). Fourteen newborns (7 hypoxia and 7 normoxia group) were scanned longitudinally to characterize physiological and morphological alterations, and axonal myelination and injury using in vivo DTI, T2 mapping, and T2-weighted MRI. Sixteen fetuses (8 hypoxia and 8 normoxia) were studied ex vivo to assess hypoxia-induced neuronal injury/loss using Nissl staining and quantitative reverse transcriptase Polymerase Chain Reaction methods.ResultsDevelopmental brains in the hypoxia group showed lower fractional anisotropy in the corpus callosum (\ue2\u2c6\u201912%, p=0.02) and lower T2 values in the hippocampus (\ue2\u2c6\u201916%, p=0.003) compared with the normoxia group with no differences in the cortex (p>0.07), indicating vulnerability of the hippocampus and cerebral white matter during early development. Fetal guinea pig brains with chronic hypoxia demonstrated an over-tenfold increase in expression levels of hypoxia index genes such as erythropoietin and HIF-1\uce\ub1, and an over 40% reduction in neuronal density, confirming prenatal brain damage.ConclusionIn vivo MRI measurement, such as DTI and T2 mapping, provides quantitative parameters to characterize neuro-developmental abnormalities and to monitor the impact of prenatal insult on the postnatal brain maturation of guinea pigs.DP00187-5/DP/NCCDPHP CDC HHS/United StatesP30 AG035982/AG/NIA NIH HHS/United StatesP30 AG035982/AG/NIA NIH HHS/United StatesP30 HD002528/HD/NICHD NIH HHS/United StatesP30 HD002528/HD/NICHD NIH HHS/United StatesR01 HL049041/HL/NHLBI NIH HHS/United StatesR01 HL049041-13/HL/NHLBI NIH HHS/United StatesR03 HD062734/HD/NICHD NIH HHS/United StatesR03 HD062734/HD/NICHD NIH HHS/United StatesS10 RR029577/RR/NCRR NIH HHS/United StatesS10 RR29577/RR/NCRR NIH HHS/United StatesUL1 RR033179/RR/NCRR NIH HHS/United StatesUL1RR033179/RR/NCRR NIH HHS/United States2016-09-01T00:00:00Z25504885PMC446805

Early-life adversity and neurological disease: age-old questions and novel answers.

Neurological illnesses, including cognitive impairment, memory decline and dementia, affect over 50 million people worldwide, imposing a substantial burden on individuals and society. These disorders arise from a combination of genetic, environmental and experiential factors, with the latter two factors having the greatest impact during sensitive periods in development. In this Review, we focus on the contribution of adverse early-life experiences to aberrant brain maturation, which might underlie vulnerability to cognitive brain disorders. Specifically, we draw on recent robust discoveries from diverse disciplines, encompassing human studies and experimental models. These discoveries suggest that early-life adversity, especially in the perinatal period, influences the maturation of brain circuits involved in cognition. Importantly, new findings suggest that fragmented and unpredictable environmental and parental signals comprise a novel potent type of adversity, which contributes to subsequent vulnerabilities to cognitive illnesses via mechanisms involving disordered maturation of brain 'wiring'

The Claustrum and Insula in Microcebus murinus: A High Resolution Diffusion Imaging Study

The claustrum and the insula are closely juxtaposed in the brain of the prosimian primate, the gray mouse lemur (Microcebus murinus). Whether the claustrum has closer affinities with the cortex or the striatum has been debated for many decades. Our observation of histological sections from primate brains and genomic data in the mouse suggest former. Given this, the present study compares the connections of the two structures in Microcebus using high angular resolution diffusion imaging (HARDI, with 72 directions), with a very small voxel size (90 micra), and probabilistic fiber tractography. High angular and spatial resolution diffusion imaging is non-destructive, requires no surgical interventions, and the connection of each and every voxel can be mapped, whereas in conventional tract tracer studies only a few specific injection sites can be assayed. Our data indicate that despite the high genetic and spatial affinities between the two structures, their connectivity patterns are very different. The claustrum connects with many cortical areas and the olfactory bulb; its strongest probabilistic connections are with the entorhinal cortex, suggesting that the claustrum may have a role in spatial memory and navigation. By contrast, the insula connects with many subcortical areas, including the brainstem and thalamic structures involved in taste and visceral feelings. Overall, the connections of the Microcebus claustrum and insula are similar to those of the rodents, cat, macaque, and human, validating our results. The insula in the Microcebus connects with the dorsolateral frontal cortex in contrast to the mouse insula, which has stronger connections with the ventromedial frontal lobe, yet this is consistent with the dorsolateral expansion of the frontal cortex in primates. In addition to revealing the connectivity patterns of the Microcebus brain, our study demonstrates that HARDI, at high resolutions, can be a valuable tool for mapping fiber pathways for multiple sites in fixed brains in rare and difficult-to-obtain species

Caffeine Combined with Sedative/Anesthetic Drugs Used in Neonatal Medicine and Apoptotic Neurotoxicity in Developing Mouse Brain

Each year, millions of premature babies are exposed to sedative/anesthetic drugs (SADs) in the neonatal intensive care unit (NICU). Acute exposure to SADs triggers widespread apoptosis in the developing brain of rodents and non-human primates. Furthermore, premature infants are administered caffeine (CAF) to treat respiratory dysfunction. Mounting evidence suggests that CAF may be neurotoxic and, when given in combination with SADs, potentiates SAD-induced cell death. However, the apoptotic interaction of CAF and SAD co-exposure is poorly understood. In a series of studies, I report that CAF combined with the NICU SADs midazolam, ketamine, or fentanyl is more neurotoxic to the postnatal day 3 (PND3) mouse brain than either CAF or SAD alone. Since many premature infants are given CAF + SADs chronically, I tested whether multiple exposures of CAF + SAD at PND3 + PND6 is more detrimental to the developing brain than a single exposure at PND6. My results indicate that the PND6 mouse brain is as vulnerable to multiple exposures of CAF + SAD on PND3 and PND6 as it is to a single exposure on PND6. Based on these results, neonatologists should exercise caution by limiting CAF + SADs co-exposure to durations necessary to ensure the survival of babies born prematurely

Registration and Analysis of Developmental Image Sequences

Mapping images into the same anatomical coordinate system via image registration is a fundamental step when studying physiological processes, such as brain development. Standard registration methods are applicable when biological structures are mapped to the same anatomy and their appearance remains constant across the images or changes spatially uniformly. However, image sequences of animal or human development often do not follow these assumptions, and thus standard registration methods are unsuited for their analysis. In response, this dissertation tackles the problems of i) registering developmental image sequences with spatially non-uniform appearance change and ii) reconstructing a coherent 3D volume from serially sectioned images with non-matching anatomies between the sections. There are three major contributions presented in this dissertation. First, I develop a similarity metric that incorporates a time-dependent appearance model into the registration framework. The proposed metric allows for longitudinal image registration in the presence of spatially non-uniform appearance change over time—a common medical imaging problem for longitudinal magnetic resonance images of the neonatal brain. Next, a method is introduced for registering longitudinal developmental datasets with missing time points using an appearance atlas built from a population. The proposed method is applied to a longitudinal study of young macaque monkeys with incomplete image sequences. The final contribution is a template-free registration method to reconstruct images of serially sectioned biological samples into a coherent 3D volume. The method is applied to confocal fluorescence microscopy images of serially sectioned embryonic mouse brains.Doctor of Philosoph

Maternal depression during early childhood, persistent aggression into emerging adulthood: neurodevelopmental pathways of risk?

Despite an accumulation of evidence documenting prospective links between maternal depression and aggression in offspring, the mechanisms underlying this association remain somewhat mysterious. Mothers' depressive symptoms could undermine offspring's learning of stage-adaptive emotion regulation (ER) skills during early childhood (e.g., Seifer, Schiller, Sameroff, Resnick, & Riordan, 1996; Silk, Shaw, Skuban, Oland, & Kovacs, 2006). Some longitudinal studies link maternal depression to disruptions in young children's ER, which has been found to predict elevated aggressive behavior in later childhood and emerging adolescence (e.g., Gilliom et al., 2002; Trentacosta & Shaw, 2009). Neurodevelopmental mechanisms such as altered organization or refinement in cortico-limbic pathways could also play a role in prospective associations between mothers' depression during early childhood and dysregulated aggression in offspring (Callaghan & Tottenham, 2016; Sheikh et al., 2014). To further inform future inquiries into these mechanisms of risk, the present study tested whether maternal depression in early childhood was prospectively linked to persistent patterns of aggression at school entry and in emerging adulthood via disruptions in early ER processes and related patterns of neuroanatomical connectivity. Participants were drawn from a sample of 310 males at elevated risk for disruptive behavior problems based on their gender and low socioeconomic status. Direct paths from maternal depression and preschool-age ER in early childhood to offspring aggression at school-age were supported. Unexpectedly, aggressive behavior was not found to be stable from the early school-age period into young adulthood across informant and context. Children's aggressive behavior was inversely associated with uncinate fasciculus structural integrity in emerging adulthood, such that higher aggression at school-age predicted lower fractional anisotropy at age 20. Another index of uncinate structural integrity (i.e., mean diffusivity) was positively associated with general antisocial behavior and depressive symptoms in young adulthood. The present findings add new, longitudinal evidence to inform nascent theories for neurodevelopmental mechanisms underlying antisocial behavior and clarify directions for future research endeavors to illuminate other potential neurodevelopmental mechanisms of risk related to mothers' depression

Role of Histone Methylation in Cognition and Effects of Different Durations of Environmental Enrichment on Learning and Memory

Part 1 Formation of stable long-term memories requires novel gene expression, and chromatin modifications have been shown to be involved in mediating the de novo gene expression which is necessary for memory formation. Although the role of histone acetylation and different histone deacetylases (HDACs) and histone acetyltransferases (HATs) in learning and memory has been studied quite extensively, the knowledge concerning the role of histone methylation – another important histone modification – in learning and memory is lacking. I have characterized the transgenic mouse line in which the Mll2 gene is conditionally knocked down at adult stage in the forebrain excitatory neurons by the Cre-LoxP system, with Cre recombinase being expressed under CamKII promoter in order to ensure recombination specifically in forebrain glutamatergic neurons knock-out mice being denoted as “F/F CKI