Abnormal Cortical Development Research Articles

Choroid plexus defects in Down syndrome brain organoids enhance neurotropism of SARS-CoV-2.

Why individuals with Down syndrome (DS) are more susceptible to SARS-CoV-2-induced neuropathology remains elusive. Choroid plexus (ChP) plays critical roles in barrier function and immune response modulation and expresses the ACE2 receptor and the chromosome 21-encoded TMPRSS2 protease, suggesting its substantial role in establishing SARS-CoV-2 infection in the brain. To explore this, we established brain organoids from DS and isogenic euploid iPSC that consist of a core of functional cortical neurons surrounded by a functional ChP-like epithelium (ChPCOs). DS-ChPCOs recapitulated abnormal DS cortical development and revealed defects in ciliogenesis and epithelial cell polarity in ChP-like epithelium. We then demonstrated that the ChP-like epithelium facilitates infection and replication of SARS-CoV-2 in cortical neurons and that this is increased in DS. Inhibiting TMPRSS2 and furin activity reduced viral replication in DS-ChPCOs to euploid levels. This model enables dissection of the role of ChP in neurotropic virus infection and euploid forebrain development and permits screening of therapeutics for SARS-CoV-2-induced neuropathogenesis.

Gray matter correlates of attention-deficit hyperactivity disorder in boys versus girls with sensory processing dysfunction

Abstract Neuroimaging shows volumetric alterations of gray matter in attention-deficit hyperactivity disorder (ADHD); however, results are conflicting. This may be due to heterogeneous phenotypic sampling and limited sensitivity of volumetric analysis. Creating more homogenous cohorts and investigating gray matter microstructure may yield meaningful biomarkers for scientific and clinical applications. Children with sensory processing dysfunction (SPD) have differences in white matter microstructure, but not gray matter volumetric differences. Approximately 40% of SPD children meet research criteria for ADHD. We apply deep learning segmentation of MRI to measure gray matter volume (GMV) and density (GMD) in SPD children with (SPD+ADHD) and without co-morbid ADHD (SPD-ADHD). We hypothesize GMV and GMD are linked to ADHD but with sex-specific regional patterns. We find boys with SPD+ADHD have widespread reduction of GMD in neocortex, limbic cortex, and cerebellum versus boys with SPD-ADHD. The greatest differences are in sensory cortex with less involvement of prefrontal regions associated with attention networks and impulse control. In contrast, changes of ADHD in girls with SPD are in brainstem nuclei responsible for dopaminergic, noradrenergic, and serotonergic neurotransmission. Hence, neural correlates of ADHD in SPD are sexually dimorphic. In boys, ADHD may result from downstream effects of abnormal sensory cortical development.

Specific inhibitor of Wnt/beta-catenin pathway can alter behavioral responses in young rats with malformed cortices

The Wnt/beta-catenin pathway plays a crucial role in regulating cellular processes and has been implicated in neural activity-dependent learning as well as anxiety. However, the role of this pathway in young children with abnormal cortical development is unknown. Cortical malformations at early development, behavioral abnormalities, and a susceptibility to seizures have been reported in rats prenatally exposed to methylazoxymethanol. In this study, we aimed to investigate whether we could improve the behavioral deficits in young rats with malformed cerebral cortices by modulation of the Wnt/beta-catenin pathway. We found a small molecule Wnt/beta-catenin inhibitor (CWP) that increased exploratory behavior in the open field test (P9, CWP 100 ug treatment, peripheral exploration, P = 0.011) and social behavior test (P12, CWP 250 ug treatment, distance traveled in center, P = 0.033) and decreased anxiety in fear conditioning. However, it did not reduce the susceptibility to seizures. After high dose (250 ug) CWP treatment at P12, phosphocreatine and glutathione (GSH) were decreased in the cortex at P15 (P = 0.021). These findings suggest that the role of Wnt/beta-catenin signaling in exploratory behavior and anxiety during early development warrants further investigation.

Neuroimaging Findings in Fetal Hemimegalencephaly: Case Study and Review.

Limited information exists in the prenatal literature regarding the neuroimaging features of fetal hemimegalencephaly. This report describes ultrasound and magnetic resonance imaging (MRI) findings in a second-trimester fetus with an isolated, severe form of hemimegalencephaly. The most prominent imaging findings included unilateral enlarged cerebral hemisphere and ipsilateral ventriculomegaly causing cerebral asymmetry, midline shift, and macrocephaly. Abnormal cortical development imaging signs were also evident. A literature review encompassing 23 reports describing 36 cases, including ours, is presented. Characteristic ultrasound findings for the diagnosis of hemimegalencephaly are not always apparent prenatally. Asymmetric ventriculomegaly emerges as the most common but nonspecific presenting feature during routine second- or third-trimester ultrasound scans. Subsequent high-resolution prenatal neurosonography and fetal MRI facilitate definitive prenatal diagnosis, showcasting associated features primarily related to cortical migration, differentiation, and maturation. Postnatally, the prognosis is poor due to intractable seizures, hemiplegia, and progressive neurodevelopmental delay.

High-resolution cortical MAP-MRI reveals areal borders and laminar substructures observed with histological staining

The variations in cellular composition and tissue architecture measured with histology provide the biological basis for partitioning the brain into distinct cytoarchitectonic areas and for characterizing neuropathological tissue alterations. Clearly, there is an urgent need to develop whole-brain neuroradiological methods that can assess cortical cyto- and myeloarchitectonic features non-invasively. Mean apparent propagator (MAP) MRI is a clinically feasible diffusion MRI method that quantifies efficiently and comprehensively the net microscopic displacements of water molecules diffusing in tissues. We investigate the sensitivity of high-resolution MAP-MRI to detecting areal and laminar variations in cortical cytoarchitecture and compare our results with observations from corresponding histological sections in the entire brain of a rhesus macaque monkey. High-resolution images of MAP-derived parameters, in particular the propagator anisotropy (PA), non-gaussianity (NG), and the return-to-axis probability (RTAP) reveal cortical area-specific lamination patterns in good agreement with the corresponding histological stained sections. In a few regions, the MAP parameters provide superior contrast to the five histological stains used in this study, delineating more clearly boundaries and transition regions between cortical areas and laminar substructures. Throughout the cortex, various MAP parameters can be used to delineate transition regions between specific cortical areas observed with histology and to refine areal boundaries estimated using atlas registration-based cortical parcellation. Using surface-based analysis of MAP parameters we quantify the cortical depth dependence of diffusion propagators in multiple regions-of-interest in a consistent and rigorous manner that is largely independent of the cortical folding geometry. The ability to assess cortical cytoarchitectonic features efficiently and non-invasively, its clinical feasibility, and translatability make high-resolution MAP-MRI a promising 3D imaging tool for studying whole-brain cortical organization, characterizing abnormal cortical development, improving early diagnosis of neurodegenerative diseases, identifying targets for biopsies, and complementing neuropathological investigations.

Infection of the murine placenta by Listeria monocytogenes induces sex-specific responses in the fetal brain.

Epidemiological data indicate that prenatal infection is associated with an increased risk of several neurodevelopmental disorders in the progeny. These disorders display sex differences in presentation. The role of the placenta in the sex-specificity of infection-induced neurodevelopmental abnormalities is not well-defined. We used an imaging-based animal model of the bacterial pathogen Listeria monocytogenes to identify sex-specific effects of placental infection on neurodevelopment of the fetus. Pregnant CD1 mice were infected with a bioluminescent strain of Listeria on embryonic day 14.5 (E14.5). Excised fetuses were imaged on E18.5 to identify the infected placentas. The associated fetal brains were analyzed for gene expression and altered brain structure due to infection. The behavior of adult offspring affected by prenatal Listeria infection was analyzed. Placental infection induced sex-specific alteration of gene expression patterns in the fetal brain and resulted in abnormal cortical development correlated with placental infection levels. Furthermore, male offspring exhibited abnormal social interaction, whereas females exhibited elevated anxiety. Placental infection by Listeria induced sex-specific abnormalities in neurodevelopment of the fetus. Prenatal infection also affected the behavior of the offspring in a sex-specific manner. Placental infection with Listeria monocytogenes induces sexually dichotomous gene expression patterns in the fetal brains of mice. Abnormal cortical lamination is correlated with placental infection levels. Placental infection results in autism-related behavior in male offspring and heightened anxiety levels in female offspring.

Schizophrenia Polygenic Risk During Typical Development Reflects Multiscale Cortical Organization

BackgroundSchizophrenia is widely recognized as a neurodevelopmental disorder. Abnormal cortical development in otherwise typically developing children and adolescents may be revealed using polygenic risk scores for schizophrenia (PRS-SCZ). MethodsWe assessed PRS-SCZ and cortical morphometry in typically developing children and adolescents (3–21 years, 46.8% female) using whole-genome genotyping and T1-weighted magnetic resonance imaging (n = 390) from the PING (Pediatric Imaging, Neurocognition, and Genetics) cohort. We contextualized the findings using 1) age-matched transcriptomics, 2) histologically defined cytoarchitectural types and functionally defined networks, and 3) case-control differences of schizophrenia and other major psychiatric disorders derived from meta-analytic data of 6 ENIGMA (Enhancing Neuro Imaging Genetics through Meta Analysis) working groups, including a total of 12,876 patients and 15,670 control participants. ResultsHigher PRS-SCZ was associated with greater cortical thickness, which was most prominent in areas with heightened gene expression of dendrites and synapses. PRS-SCZ–related increases in vertexwise cortical thickness were mainly distributed in association cortical areas, particularly the ventral attention network, while relatively sparing koniocortical type cortex (i.e., primary sensory areas). The large-scale pattern of cortical thickness increases related to PRS-SCZ mirrored the pattern of cortical thinning in schizophrenia and mood-related psychiatric disorders derived from the ENIGMA consortium. Age group models illustrate a possible trajectory from PRS-SCZ–associated cortical thickness increases in early childhood toward thinning in late adolescence, with the latter resembling the adult brain phenotype of schizophrenia. ConclusionsCollectively, combining imaging genetics with multiscale mapping, our work provides novel insight into how genetic risk for schizophrenia affects the cortex early in life.

A rare case of hemimegalencephaly diagnosed prenatally

Hemimegalencephaly (HME), or unilateral megalencephaly, is a rare congenital brain malformation defined as overgrowth of one cerebral hemisphere or part of it resulting from abnormal cortical development and neuronal migration. However, cortical developmental abnormalities are rarely diagnosed prenatally. This is the reason for our study, in which we describe and compare ultrasound and MRI findings in a fetus with HME.

Blood brain barrier as an interface for alcohol induced neurotoxicity during development

Ethanol consumption during pregnancy or lactation permanently impairs the development of the central nervous system (CNS), resulting in the spectrum of fetal alcohol disorders (FASD). FASD is a general term that covers a set of deficits in the embryo caused by gestational alcohol exposure, with fetal alcohol syndrome (FAS) considered the most serious. The clinical features of FAS include facial abnormalities, short stature, low body weight, and evidence of structural and/or functional damage to the central nervous system (CNS). The prevalence of FAS carriers worldwide is about 15 for every 10,000 live births (about 119,000 children with APS born per year). Epidemiological data in the US show that the incidence of FAS exceeds other congenital syndromes such as Down syndrome and spina bifida. The deleterious effects of ethanol appear in different brain regions, varying according to the dose and period of neural development when the embryo was exposed, and include: 1) microcephaly; 2) abnormalities in cortical development, with a significant decrease in gyrification; 3) agenesis or hypoplasia of the corpus callosum; and 4) cognitive and behavioral deficits (such as impaired memory and learning, speech difficulties, and hyperactivity). Current evidence indicates that CNS blood vessels are particularly affected by teratogenic ethanol. The CNS vasculature is composed of specialized endothelial cells that establish intimate interactions with astrocytes, pericytes, and microglia, constituting the neurovascular unit of the blood-brain barrier (BBB). Together with the fact that BBB exert protective function, it can prevent the passage of substances and drugs to treat diseases that affect the CNS. Pathological changes in the BBB, such as drug abuse during pregnancy, congenital infections, or ageing processes can drastically alter the molecular structure and vascular stability, disrupting the BBB and aggravating certain neurodegenerative and neurological diseases. In this review, we address the effects of alcohol exposure on the formation of the BBB, specifically describing the cellular and molecular events induced by ethanol in the physiology of endothelial cells and glial cells, as well as their interaction during CNS development.

Immunohistochemical analysis of the developing mouse cortex.

The cerebral cortex is the most complex structure in the mammalian brain, whose development requires coordinated proliferation of neural stem/precursor cells (NPCs) and their differentiation into neurons and glia. Perturbations in NPC homeostasis can lead to abnormal cortical development which is frequently seen in neurodevelopmental disorders. In this chapter, we describe the preparation of cortical tissues from mice and step-by-step protocol for immunohistochemistry to study cortical development. With this technique, we employ commonly used molecular markers and thymidine analog methods to analyze NPC populations. We also discuss assay conditions that can be optimized according to the specific needs to improve experimental outcomes.

KIF5C deficiency causes abnormal cortical neuronal migration, dendritic branching, and spine morphology in mice.

Malformation of cortical development (MCD) includes a variety of developmental disorders that are common causes of neurodevelopmental delay and epilepsy. Most recently, clinical studies found that patients carrying KIF5C mutations present early-onset MCD; however, the underlying mechanisms remain elusive. KIF5C expression level was examined in mouse primary cortical neurons and human ips-derived forebrain organoids. We studied the cortical neuronal migration, dendritic branching, and dendritic spine growth after knocking down the KIF5C gene by electroporation in vitro and in vivo. Then, we studied the transcriptome differences between the knockdown and control groups through RNA sequencing. We observed high KIF5C expression in neurons during the early developmental stage in mice and the human brain. Kif5c deficiency results in disturbed cortical neuronal migration, dendritic, and spine growth. Finally, we found that Kif5c knockdown affected several genes associated with cortical neuronal development in vitro. These results suggested a critical role for Kif5c in cortical development, providing insights into underlying pathogenic factors of kinesins in MCD. KIF5C mutation-related MCD might be caused by abnormal early cortical neuronal development. Kif5c deficiency led to abnormal cortical neuronal dendritic and spine growth and neuronal migration. Our findings explain how Kif5c deficiency is involved in the aberrant development of cortical neurons and provide a new perspective for the pathology of MCD.

SNAP-25 Single Nucleotide Polymorphisms, Brain Morphology and Intelligence in Children With Borderline Intellectual Functioning: A Mediation Analysis.

Borderline intellectual functioning (BIF) is a multifactorial condition in which both genetic and environmental factors are likely to contribute to the clinical outcome. Abnormal cortical development and lower IQ scores were shown to be correlated in BIF children, but the genetic components of this condition and their possible connection with intelligence and brain morphology have never been investigated in BIF. The synaptosomal-associated protein of 25 kD (SNAP-25) is involved in synaptic plasticity, neural maturation, and neurotransmission, affecting intellectual functioning. We investigated SNAP-25 polymorphisms in BIF and correlated such polymorphisms with intelligence and cortical thickness, using socioeconomic status and environmental stress as covariates as a good proxy of the variables that determine intellectual abilities. Thirty-three children with a diagnosis of BIF were enrolled in the study. SNAP-25 polymorphisms rs363050, rs363039, rs363043, rs3746544, and rs1051312 were analyzed by genotyping; cortical thickness was studied by MRI; intelligence was measured using the WISC-III/IV subscales; environmental stressors playing a role in neuropsychiatric development were considered as covariate factors. Results showed that BIF children carrying the rs363043(T) minor allele represented by (CT + TT) genotypes were characterized by lower performance Perceptual Reasoning Index and lower full-scale IQ scores (p = 0.04) compared to those carrying the (CC) genotype. This association was correlated with a reduced thickness of the left inferior parietal cortex (direct effect = 0.44) and of the left supramarginal gyrus (direct effect = 0.56). These results suggest a link between SNAP-25 polymorphism and intelligence with the mediation role of brain morphological features in children with BIF.

Cortical thickness in clinical moyamoya disease: A magnetic resonance imaging study.

Moyamoya disease (MMD) is a progressive cerebrovascular disorder, with an unknown pathogenesis and aetiology. MMD is characterized by steno-occlusive changes at the terminal portion of the internal carotid artery (ICA), which is accompanied by variable development of the basal collaterals, also known as moyamoya vessels. Patients with MMD show variable patterns of brain damage and may experience recurrent multiple transient ischaemic attacks, intracranial bleeding and cerebral infarction. In this study, we investigate the potential for structural T1 magnetic resonance imaging (MRI) to help characterize abnormal cortical development in MMD clinically, with an analysis of both average and variability of regional cortical thicknesses. This study also included a machine learning analysis to assess the predictive capacity of the cortical thickness abnormalities observed in this research. This study included 993 MRI examinations from neurotypical controls and 269 MRI examinations from MMD patients. Results demonstrate abnormal cortical presentation of the insula, caudate, postcentral, precuneus and cingulate regions, in agreement with previous literature cortical thickness findings as well as alternative methods such as functional MRI (fMRI) and digital angiography. To the best of our knowledge, this is the first manuscript to report cortical thickness abnormalities in the middle temporal visual area in MMD and the first study to report on cortical thickness variability abnormalities in MMD.

Neuroimaging in Children Born With Congenital Zika Syndrome: A Cohort Study

Neuroimaging findings have been associated with adverse neurologic outcomes in children with congenital Zika virus infection. Our purpose is to describe the brain magnetic resonance imaging (MRI) of children around 3 years of age, born with congenital Zika syndrome. This cohort study followed 62 children born with congenital Zika syndrome who had head computed tomography (CT) performed during the first months of life. All these children had clinical, neuroimaging, and serological confirmation of congenital Zika. Around 3 years of age, these children received a brain MRI. In 35 children, we could perform an MRI. All these children had severe impairment in neuromotor development. In general, the examinations showed the same alterations of the CT examinations: delayed myelination (82.8%), intracranial calcification (71.4%) although with decreased intensity and size as compared with previously CT examinations, ventriculomegaly (91.4%), cerebellar hypoplasia (68.5%), and cortical development abnormalities (85.8%). The serious brain alterations observed through head CT examinations in children born with congenital Zika syndrome continued to be detected through an MRI examination carried out at around 3 years of age. This indicates a poor prognosis for these children who had a severe neuromotor development delay.

STRADA-mutant human cortical organoids model megalencephaly and exhibit delayed neuronal differentiation.

Genetic diseases involving overactivation of the mechanistic target of rapamycin (mTOR) pathway, so-called "mTORopathies," often manifest with malformations of cortical development (MCDs), epilepsy, and cognitive impairment. How mTOR pathway hyperactivation results in abnormal human cortical development is poorly understood. To study the effect of mTOR hyperactivity on early stages of cortical development, we focused on Pretzel Syndrome (polyhydramnios, megalencephaly, symptomatic epilepsy; PMSE syndrome), a rare mTORopathy caused by homozygous germline mutations in the STRADA gene. We developed a human cortical organoid (hCO) model of PMSE and examined morphology and size for the first 2weeks of organoid growth, and cell type composition at weeks 2, 8, and 12 of differentiation. In the second week, PMSE hCOs enlarged more rapidly than controls and displayed an abnormal Wnt pathway-dependent increase in neural rosette structures. PMSE hCOs also exhibited delayed neurogenesis, decreased subventricular zone progenitors, increased proliferation and cell death, and an abnormal architecture of primary cilia. At week 8, PMSE hCOs had fewer deep layer neurons. By week 12, neurogenesis recovered in PMSE organoids, but they displayed increased outer radial glia, a cell type thought to contribute to the expansion of the human cerebral cortex. Together, these findings suggest that megalencephaly in PMSE arises from the expansion of neural stem cells in early corticogenesis and potentially also from increased outer radial glial at later gestational stages. The delayed neuronal differentiation in PMSE organoids demonstrates the important role the mTOR pathway plays in the maintenance and expansion of the stem cell pool.

Association between Quantitative MR Markers of Cortical Evolving Organization and Gene Expression during Human Prenatal Brain Development.

The relationship between structural changes of the cerebral cortex revealed by Magnetic Resonance Imaging (MRI) and gene expression in the human fetal brain has not been explored. In this study, we aimed to test the hypothesis that relative regional thickness (a measure of cortical evolving organization) of fetal cortical compartments (cortical plate [CP] and subplate [SP]) is associated with expression levels of genes with known cortical phenotype. Mean regional SP/CP thickness ratios across age measured on in utero MRI of 25 healthy fetuses (20-33 gestational weeks [GWs]) were correlated with publicly available regional gene expression levels (23-24 GW fetuses). Larger SP/CP thickness ratios (more pronounced cortical evolving organization) was found in perisylvian regions. Furthermore, we found a significant association between SP/CP thickness ratio and expression levels of the FLNA gene (mutated in periventricular heterotopia, congenital heart disease, and vascular malformations). Further work is needed to identify early MRI biomarkers of gene expression that lead to abnormal cortical development.

ATF5 deficiency causes abnormal cortical development

Activating transcription factor 5 (ATF5) is a member of the cAMP response element binding protein (CREB)/ATF family of basic leucine zipper transcription factors. We previously reported that ATF5-deficient (ATF5−/−) mice exhibited behavioural abnormalities, including abnormal social interactions, reduced behavioural flexibility, increased anxiety-like behaviours, and hyperactivity in novel environments. ATF5−/− mice may therefore be a useful animal model for psychiatric disorders. ATF5 is highly expressed in the ventricular zone and subventricular zone during cortical development, but its physiological role in higher-order brain structures remains unknown. To investigate the cause of abnormal behaviours exhibited by ATF5−/− mice, we analysed the embryonic cerebral cortex of ATF5−/− mice. The ATF5−/− embryonic cerebral cortex was slightly thinner and had reduced numbers of radial glial cells and neural progenitor cells, compared to a wild-type cerebral cortex. ATF5 deficiency also affected the basal processes of radial glial cells, which serve as a scaffold for radial migration during cortical development. Further, the radial migration of cortical upper layer neurons was impaired in ATF5−/− mice. These results suggest that ATF5 deficiency affects cortical development and radial migration, which may partly contribute to the observed abnormal behaviours.

Abnormal Left-Hemispheric Sulcal Patterns in Adults With Simple Congenital Heart Defects Repaired in Childhood.

BackgroundChildren operated on for a simple congenital heart defect (CHD) are at risk of neurodevelopmental abnormalities. Abnormal cortical development and folding have been observed in fetuses with CHD. We examined whether sulcal folding patterns in adults operated on for simple CHD in childhood differ from those of healthy controls, and whether such differences are associated with neuropsychological outcomes.Methods and ResultsPatients (mean age, 24.5 years) who underwent childhood surgery for isolated atrial septal defect (ASD; n=33) or ventricular septal defect (VSD; n=30) and healthy controls (n=37) were enrolled. Sulcal pattern similarity to healthy controls was determined using magnetic resonance imaging and looking at features of sulcal folds, their intersulcal relationships, and sulcal graph topology. The sulcal pattern similarity values were tested for associations with comprehensive neuropsychological scores. Patients with both ASD and VSD had decreased sulcal pattern similarity in the left hemisphere compared with controls. The differences were found in the left temporal lobe in the ASD group and in the whole left hemisphere in the VSD group (P=0.033 and P=0.039, respectively). The extent of abnormal left hemispheric sulcal pattern similarity was associated with worse neuropsychological scores (intelligence, executive function, and visuospatial abilities) in the VSD group, and special educational support in the ASD group.ConclusionsAdults who underwent surgery for simple CHD in childhood display altered left hemisphere sulcal folding patterns, commensurate with neuropsychological scores for patients with VSD and special educational support for ASD. This may indicate that simple CHD affects early brain development.RegistrationURL: https://www.clinicaltrials.gov; Unique identifier: NCT03871881.

Abnormal Rat Cortical Development Induced by Ventricular Injection of rHMGB1 Mimics the Pathophysiology of Human Cortical Dysplasia.

Cortical dysplasia (CD) is a common cause of drug-resistant epilepsy. Increasing studies have implicated innate immunity in CD with epilepsy. However, it is unclear whether innate immune factors induce epileptogenic CD. Here, we injected recombinant human high mobility group box 1 (rHMGB1) into embryonic rat ventricles to determine whether rHMGB1 can induce epileptogenic CD with pathophysiological characteristics similar to those of human CD. Compared with controls and 0.1 μg rHMGB1-treated rats, the cortical organization was severely disrupted in the 0.2 μg rHMGB1-treated rats, and microgyria and heterotopia also emerged; additionally, disoriented and deformed neurons were observed in the cortical lesions and heterotopias. Subcortical heterotopia appeared in the white matter and the gray–white junction of the 0.2 μg rHMGB1-treated rats. Moreover, there was decreased number of neurons in layer V–VI and an increased number of astrocytes in layer I and V of the cortical lesions. And the HMGB1 antagonist dexmedetomidine alleviated the changes induced by rHMGB1. Further, we found that TLR4 and NF-κB were increased after rHMGB1 administration. In addition, the excitatory receptors, N-methyl-D-aspartate receptor 1 (NR1), 2A (NR2A), and 2B (NR2B) immunoreactivity were increased, and immunoreactivity of excitatory amino acid transporter 1 (EAAT1) and 2 (EAAT2) were reduced in 0.2 μg rHMGB1-treated rats compared with controls. While there were no differences in the glutamic acid decarboxylase 65/67 (GAD65/67) immunoreactivity between the two groups. These results indicate that the excitation of cortical lesions was significantly increased. Furthermore, electroencephalogram (EEG) showed a shorter latency of seizure onset and a higher incidence of status epilepticus in the 0.2 μg rHMGB1-treated rats; the frequency and amplitude of EEG were higher in the treated rats than controls. Intriguingly, spontaneous electrographic seizure discharges were detected in the 0.2 μg rHMGB1-treated rats after 5 months of age, and spike-wave discharges of approximately 8 Hz were the most significantly increased synchronous propagated waves throughout the general brain cortex. Taken together, these findings indicate that rHMGB1 exposure during pregnancy could contribute to the development of epileptogenic CD, which mimicked some pathophysiological characteristics of human CD.

The zinc transporter ZIP9 (Slc39a9) regulates zinc dynamics essential to egg activation in zebrafish

The zinc transporter ZIP9 (SLC39A9) was recently characterized as a membrane androgen receptor in various teleost and mammalian cell models. ZIP9 shows the highest expression in ovaries of teleosts, a tissue in which both androgen signaling and zinc dynamics have significant roles. To examine the role of ZIP9 in ovarian physiology, we generated a ZIP9-mutant zebrafish strain using a CRISPR/Cas9 system. zip9-/- females showed significant reductions in fecundity, embryo viability, and growth of their offspring compared to wildtype (WT) fish. Furthermore, a high proportion of zip9-/- eggs failed to undergo normal chorion elevation during activation. In WT eggs, zinc was detected in cortically-localized vesicles which underwent exocytosis upon activation. zip9-/- eggs showed abnormal cortical vesicle development and had a significantly depressed activation-induced zinc release compared to WT eggs. Moreover, pharmacologically sustained elevation of zinc in WT eggs prior to activation resulted in abnormal chorion elevation similar to that observed in zip9-/- eggs. These results indicate that ZIP9 is essential for proper zinc modulation during zebrafish egg activation and presents the first evidence of zinc modulation during egg activation in a non-mammalian species.