Prenatal Mouse Research Articles

Assessing the Expression of Major Histocompatibility Complex Class I on Primary Murine Hippocampal Neurons by Flow Cytometry

Increasing evidence supports the hypothesis that neuro-immune interactions impact nervous system function in both homeostatic and pathologic conditions. A well-studied function of major histocompatibility complex class I (MHCI) is the presentation of cell-derived peptides to the adaptive immune system, particularly in response to infection. More recently it has been shown that the expression of MHCI molecules on neurons can modulate activity-dependent changes in the synaptic connectivity during normal development and neurologic disorders. The importance of these functions to the brain health supports the need for a sensitive assay that readily detects MHCI expression on neurons. Here we describe a method for primary culture of murine hippocampal neurons and then assessment of MHCI expression by flow cytometric analysis. Murine hippocampus is microdissected from prenatal mouse pups at the embryonic day 18. Tissue is dissociated into a single cell suspension using enzymatic and mechanical techniques, then cultured in a serum-free media that limits growth of non-neuronal cells. After 7 days in vitro, MHCI expression is stimulated by treating cultured cells pharmacologically with beta interferon. MHCI molecules are labeled in situ with a fluorescently tagged antibody, then cells are non-enzymatically dissociated into a single cell suspension. To confirm the neuronal identity, cells are fixed with paraformaldehyde, permeabilized, and labeled with a fluorescently tagged antibody that recognizes neuronal nuclear antigen NeuN. MHCI expression is then quantified on neurons by flow cytometric analysis. Neuronal cultures can easily be manipulated by either genetic modifications or pharmacologic interventions to test specific hypotheses. With slight modifications, these methods can be used to culture other neuronal populations or to assess expression of other proteins of interest.

Protective Effect of Astaxanthin on Prenatal Bacterial Lipopolysaccharide Exposed Behavioral Deficits in Adult Mice

Background: Prenatal maternal lipopolysaccharide (LPS) exposure causes behavioral deficits in adulthood. LPS-exposure cause oxidative damage and cytokines production. In contrast, astaxanthin (Ast) is a carotenoid antioxidant that shows protective effects through its antioxidant capacity. Objectives: This study investigates the effect of prenatal treatment with astaxanthin on the behavioral deficit (including sexual, depressive, and anxiety-like behavior) caused by prenatal maternal LPS in adult male offspring. Methods: Pregnant mice were randomly divided into 4 groups: (1) control, (2) LPS: injecting with LPS (20 µg/kg, sc.) on gestation day 11, (3) Ast: receiving astaxanthin (4 mg/kg for 3 days, i.p.) on 11 - 13th gestation day, (4) LPS+Ast: injecting with LPS (20 µg/kg, sc.) on gestation day 11 and receiving astaxanthin (4 mg/kg for 3 days, i.p.) on 11 - 13th gestation day. Then in each group, 23 day old male offspring (3 and 12 male children from each mother and group, respectively) were separated from mothers and then the sexual, depressive and anxiety-like behaviors were examined in adult male mice. Results: Findings showed that prenatal LPS-exposed mice had more anxiety and spent less time in open arms of the elevated plus-maze test (P < 0.05). In addition, it decreased sexual behaviors, the amount of which was significant in the number of sniffing, following behaviors (P < 0.01). Also, there was no significant difference between different groups in the forced swimming test (P < 0.05). On the other hand, prenatal treatment with astaxanthin significantly elevated the percentage of open arm time and open arm entry, without altering in locomotor activity (P < 0.05). Also, it significantly increased sexual behavior in Ast and LPS+Ast groups (P < 0.01). Conclusions: The obtained results suggest that prenatal maternal exposure to LPS impaired several aspects of male sexual behavior and resulted in behavioral deficits in adulthood, while astaxanthin has an antianxiety effect and improves the deficits of sexual behavior presumably via its antioxidant property.

Elucidating the role of pigment epithelium-derived factor (PEDF) in metabolic PCOS models.

PCOS is the most common endocrinopathy in women; associated with obesity and insulin resistance (IR). IR leads to accumulation of advanced-glycation-end-products (AGEs) and their receptor, RAGE. PCOS patients have increased levels of vascular endothelial growth factor (VEGF), interleukin 6/8 (IL-6/8) and anti-Mϋllerian-hormone (AMH). PEDF is a secreted-glycoprotein known for its anti-angiogenic and anti-inflammatory properties. We aimed to elucidate the role of PEDF in the pathogenesis and treatment of PCOS. We used a prenatal PCOS mouse model and fed the female offspring a high-fat diet, inducing metabolic PCOS (met.PCOS) characteristics. Female offspring were divided into three groups: control; met.PCOS; met.PCOS + recombinant PEDF (rPEDF). Met.PCOS mice gained more weight, had elevated serum IL-6 and higher mRNA levels of AMH, PEDF and RAGE in their granulosa cells (GCs) than met.PCOS + rPEDF mice. An in vitro Met.PCOS model in human GCs (KGN) line was induced by prolonged incubation with insulin/AGEs, causing development of IR. Under the same conditions, we observed an elevation of VEGF, IL-6/8 mRNAs, concomitantly with an increase in PEDF mRNA, intracellular protein levels, and an elevation of PEDF receptors (PEDF-Rs) mRNA and protein. Simultaneously, a reduction in the secretion of PEDF from GCs, was measured in the medium. The addition of rPEDF (5 nM) activated P38 signaling, implying that PEDF-Rs maintained functionality, and negated AGE-induced elevation of IL-6/8 and VEGF mRNAs. Decreased PEDF secretion may be a major contributor to hyperangiogenesis and chronic inflammation, which lie at the core of PCOS pathogenesis. rPEDF treatment may restore physiological angiogenesis inflammatory balance, thus suggesting a potential therapeutic role in PCOS.

Prenatal arsenic exposure interferes in postnatal immunocompetence despite an absence of ongoing arsenic exposure

Arsenic (As) readily crosses the placenta and exposure of the fetus may cause adverse consequences later in life, including immunomodulation. In the current study, the question was asked how the immune repertoire might respond in postnatal life when there is no further As exposure. Here, pregnant mice (Balb/c [H-2d]) were exposed to arsenic trioxide (As2O3) through their drinking water from time of conception until parturition. Their offspring, 4-week-old mice who had not been exposed again to As, were used for functional analyses of innate, humoral and cellular immunity. Compared to cells from non-As-exposed dam offspring, isolated peritoneal macro-phages (Mϕ) displayed no differences in T-cell stimulating ability. Levels of circulating IgG2a but not IgG1 were decreased in As-exposed dam offspring as compared to control offspring counterparts. Mixed-leukocyte reactions (MLR) indicated that CD4+ T-cells from the prenatal As-exposed mice were significantly less responsive to allogenic stimulation as evidenced by decreases in interferon (IFN)-γ and IL-2 production and in expression of CD44 and CD69 (but not CD25) activation markers. Interestingly, the Mϕ from the prenatal As-exposed mice were capable of stimulating normal allogenic T-cells, indicating that T-cells from these mice were refractory to allogenic signals. There was also a significant decrease in absolute numbers of splenic CD4+ and CD8+ T-cells due to prenatal As exposure (as compared to control). Lastly, the impaired immune function of the prenatal As-exposed mice was correlated with a very strong susceptibility to Escherichia coli infection. Taken together, the data from this study clearly show that in utero As exposure may continue to perpetuate a dampening effect on the immune repertoire of offspring, even into the early stages of postnatal life.

Ontogenic mRNA expression of RNA modification writers, erasers, and readers in mouse liver.

RNA modifications are recently emerged epigenetic modifications. These diverse RNA modifications have been shown to regulate multiple biological processes, including development. RNA modifications are dynamically controlled by the “writers, erasers, and readers”, where RNA modifying proteins are able to add, remove, and recognize specific chemical modification groups on RNAs. However, little is known about the ontogenic expression of these RNA modifying proteins in various organs, such as liver. In the present study, the hepatic mRNA expression of selected RNA modifying proteins involve in m6A, m1A, m5C, hm5C, m7G, and Ψ modifications was analyzed using the RNA-seq technique. Liver samples were collected from male C57BL/6 mice at several ages from prenatal through neonatal, infant, child to young adult. Results showed that most of the RNA modifying proteins were highly expressed in prenatal mouse liver with a dramatic drop at birth. After birth, most of the RNA modifying proteins showed a downregulation trend during liver maturation. Moreover, the RNA modifying proteins that belong to the same enzyme family were expressed at different abundances at the same ages in mouse liver. In conclusion, this study unveils that the mRNA expression of RNA modifying proteins follows specific ontogenic expression patterns in mice liver during maturation. These data indicated that the changes in expression of RNA modifying proteins might have a potential role to regulate gene expression in liver through alteration of RNA modification status.

EXTH-47. THERAPEUTIC REVERSAL OF PRENATAL PONTINE ID1 SIGNALING IN DIPG

Abstract Diffuse intrinsic pontine gliomas (DIPGs) are lethal brain tumors with no effective therapies other than radiation. Inhibitor of DNA binding (ID) proteins are key regulators of tissue and lineage-specific gene differentiation during embryogenesis. Previous work has shown that H3F3A and ACVR1 mutations increase ID1 expression in cultured astrocytes, but this has not been validated in human DIPG, nor has the regulation and targetability of ID1 been explored in DIPG. Analysis of multi-focal post-mortem tumor and normal brain tissue (n=52) as well as multiple human datasets revealed ID1 to be elevated in DIPG. Elevated ID1 was found to correlate with reduced survival in DIPG. In a multi-focal autopsy DIPG case, we found ID1 expression to be heterogeneous and to correlate with tumor invasion. Chromatin immunoprecipitation-qPCR (ChIP-qPCR) was used to quantify H3K27ac and H3K27me3 at ID1 gene regulatory regions (promoters/enhancers) in multi-focal post-mortem tissue. The ID1 loci was found to be epigenetically poised for up-regulation (elevated H3K27ac and low H3K27me3) in DIPG tissue, regardless of H3 or ACVR1 mutation status, compared to normal brain. Analysis of publically-available ISH and ChIP-sequencing data revealed elevated ID1 expression and ID1-enhancer H3K27ac in prenatal mouse hindbrain compared to prenatal forebrain, prenatal midbrain, and all postnatal brain regions. ID1 shRNA-mediated knockdown of primary human H3K27M DIPG cells (DIPG007) resulted in significantly reduced invasion and migration. As cannabidiol (CBD) has successfully been used to therapeutically target ID1 in pre-clinical models of adult human cancers, we treated DIPG007 cells with CBD and found reduced viability at clinically relevant dosing (IC50=2.4 uM) with dose-dependent reduction in ID1 protein. ID1 knockdown and CBD treatment studies in murine models of DIPG are ongoing. These findings indicate that a multifactorial (genetic and regionally-based) epigenetic upregulation of ID1 drives DIPG invasiveness and is targetable with CBD.

Three paternally imprinted regions are sequentially required in prenatal and postnatal mouse development.

Three paternally imprinted regions are sequentially required in prenatal and postnatal mouse development.

Chronic Activation of the Dopaminergic Neuronal Pathway Improves Behavioral Abnormalities in the Prenatal Valproic Acid Exposure Mouse Model of Autism Spectrum Disorder

Over the last decade there has been an increase in the prevalence of autism spectrum disorder (ASD); however, its pathogenic mechanisms remain unclear. To date, no effective drug has been developed to treat the core symptoms of ASD, especially social interaction deficits. Previous studies have mainly focused on the glutamatergic, GABAergic, and serotonergic signaling pathways; however, a growing number of studies have reported abnormalities in the dopaminergic pathway, such as mutations and functional alterations of dopamine-related molecules, in ASD patients. Furthermore, atypical antipsychotic drugs risperidone and aripiprazole are prescribed for the treatment of non-core symptoms, such as irritability, in patients with ASD. These observations suggest that the dopaminergic pathway is involved in the pathogenesis of ASD. Previously, we have established a mouse model of ASD based on clinical research, which shows that exposure to valproic acid, an antiepileptic drug, during pregnancy causes an increase in the risk of developing ASD in children. This review summarizes our recent studies, which have assessed alterations in the prefrontal dopaminergic pathway. In addition, we discuss the effects of treatment with attention deficit/hyperactivity disorder drugs and atypical antipsychotic drugs, which activate the prefrontal dopaminergic pathway, on ASD-like behavioral abnormalities in the valproic acid exposure mouse model of ASD.

Disruption of SynGAP-dopamine D1 receptor complexes alters actin and microtubule dynamics and impairs GABAergic interneuron migration.

Disruption of γ-aminobutyric acid (GABA)-ergic interneuron migration is implicated in various neurodevelopmental disorders, including autism spectrum disorder and schizophrenia. The dopamine D1 receptor (D1R) promotes GABAergic interneuron migration, which is disrupted in various neurological disorders, some of which are also associated with mutations in the gene encoding synaptic Ras-guanosine triphosphatase-activating protein (SynGAP). Here, we explored the mechanisms underlying these associations and their possible connection. In prenatal mouse brain tissue, we found a previously unknown interaction between the D1R and SynGAP. This D1R-SynGAP interaction facilitated D1R localization to the plasma membrane and promoted D1R-mediated downstream signaling pathways, including phosphorylation of protein kinase A and p38 mitogen-activated protein kinase. These effects were blocked by a peptide (TAT-D1Rpep) that disrupted the D1R-SynGAP interaction. Furthermore, disrupting this complex in mice during embryonic development resulted in pronounced and selective deficits in the tangential migration of GABAergic interneurons, possibly due to altered actin and microtubule dynamics. Our results provide insights into the molecular mechanisms regulating interneuron development and suggest that disruption of the D1R-SynGAP interaction may underlie SYNGAP1 mutation-related neurodevelopmental disorders.

Three-dimensional imaging of the extracellular matrix and cell interactions in the developing prenatal mouse cornea

As the outer lens in the eye, the cornea needs to be strong and transparent. These properties are governed by the arrangement of the constituent collagen fibrils, but the mechanisms of how this develops in mammals is unknown. Using novel 3-dimensional scanning and conventional transmission electron microscopy, we investigated the developing mouse cornea, focusing on the invading cells, the extracellular matrix and the collagen types deposited at different stages. Unlike the well-studied chick, the mouse cornea had no acellular primary stroma. Collagen fibrils initially deposited at E13 from the presumptive corneal stromal cells, become organised into fibril bundles orthogonally arranged between cells. Extensive cell projections branched to adjacent stromal cells and interacted with the basal lamina and collagen fibrils. Types I, II and V collagen were expressed from E12 posterior to the surface ectoderm, and became widespread from E14. Type IX collagen localised to the corneal epithelium at E14. Type VII collagen, the main constituent of anchoring filaments, was localised posterior to the basal lamina. We conclude that the cells that develop the mouse cornea do not require a primary stroma for cell migration. The cells have an elaborate communication system which we hypothesise helps cells to align collagen fibrils.

Sex Differences in Androgen Receptor Enhancer Activity and Expression during Mus musculus Embryonic Development

Study objectiveThe objective of this study is to compare the sex‐specific enhancer function of a cis‐regulatory element during mammary gland development to the expression of the androgen receptor (AR) in mammary tissues.IntroductionConserved noncoding elements regulate gene expression during mammalian development with remarkable precision. AR is critical for the development of male specific phenotypes in many tissues, yet little is known regarding the tissue‐specific regulation of AR expression. We have previously identified a 7.7kb sequence near the AR gene that activates lacZ reporter gene expression in prenatal and postnatal mice. Embryologically, the enhancer drives consistent lacZ expression in developing hair follicles and the genital tubercle. However, expression in the mammary ridge/glands occurs in only half of the transgenic embryos produced. Each of these structures are known sites of expression of AR or are affected by androgens during growth and development. We hypothesize that the variability in this enhancer function during mammary gland development may be sex dependent.MethodsWe utilized a previously established transgenic mouse line containing the 7.7kb AR enhancer mouse sequence linked to a basal promoter driving lacZ. Mice were collected and stained during different stages of embryonic development to visualize localization of AR enhancer activity. The sex of the embryos were identified by PCR amplification of the Sry gene. We compared enhancer driven lacZ expression to endogenous AR expression determined by in situ hybridization (ISH) in mouse embryos.ResultsWe have surveyed lacZ expression at E15.5 (embryonic day) and E16.5. Despite consistent expression in other tissues, mammary gland expression of lacZ occurs only in female embryos. No mammary associated lacZ expression is visible in males. These expression results are consistent with AR gene expression as detected by ISH.ConclusionWe show that there are sex differences in enhancer activity of AR during mouse development. AR is crucial for inhibiting the development of female‐specific traits as well as promoting the male phenotype. The lack of lacZ expression in male embryos suggests the inhibitory effects of androgens have been sufficient to either eliminate the mammary associated cell types that normally express AR or the expression of an upstream trans‐acting factor that activates this AR enhancer. This mouse line will be useful for tracing developmental progression of AR expressing tissues and signalling during mammalian integumentary development as well as the development of sexually dimorphic characteristics.Support or Funding InformationFunding provided by NSF IOS‐1656315 &amp; NSF BCS‐1638812 and PCOM's Center for Chronic Disorders of Aging.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mapping developmental maturation of inner hair cell ribbon synapses in the apical mouse cochlea

Ribbon synapses of cochlear inner hair cells (IHCs) undergo molecular assembly and extensive functional and structural maturation before hearing onset. Here, we characterized the nanostructure of IHC synapses from late prenatal mouse embryo stages (embryonic days 14-18) into adulthood [postnatal day (P)48] using electron microscopy and tomography as well as optical nanoscopy of apical turn organs of Corti. We find that synaptic ribbon precursors arrive at presynaptic active zones (AZs) after afferent contacts have been established. These ribbon precursors contain the proteins RIBEYE and piccolino, tether synaptic vesicles and their delivery likely involves active, microtubule-based transport pathways. Synaptic contacts undergo a maturational transformation from multiple small to one single, large AZ. This maturation is characterized by the fusion of ribbon precursors with membrane-anchored ribbons that also appear to fuse with each other. Such fusion events are most frequently encountered around P12 and hence, coincide with hearing onset in mice. Thus, these events likely underlie the morphological and functional maturation of the AZ. Moreover, the postsynaptic densities appear to undergo a similar refinement alongside presynaptic maturation. Blockwise addition of ribbon material by fusion as found during AZ maturation might represent a general mechanism for modulating ribbon size.

Shank3 Transgenic and Prenatal Zinc-Deficient Autism Mouse Models Show Convergent and Individual Alterations of Brain Structures in MRI.

Research efforts over the past decades have unraveled both genetic and environmental factors, which contribute to the development of autism spectrum disorders (ASD). It is, to date, largely unknown how different underlying causes result in a common phenotype. However, the individual course of development and the different comorbidities might reflect the heterogeneous genetic and non-genetic contributions. Therefore, it is reasonable to identify commonalities and differences in models of these disorders at the different hierarchical levels of brain function, including genetics/environment, cellular/synaptic functions, brain regions, connectivity, and behavior. To that end, we investigated Shank3 transgenic mouse lines and compared them with a prenatal zinc-deficient (PZD) mouse model of ASD at the level of brain structural alterations in an 11,7 T small animal magnetic resonance imaging (MRI). Animals were measured at 4 and 9 weeks of age. We identified a decreased total brain volume (TBV) and hippocampal size of Shank3−/− mice but a convergent increase of basal ganglia (striatum and globus pallidus) in most mouse lines. Moreover, Shank3 transgenic mice had smaller thalami, whereas PZD mice had this region enlarged. Intriguingly, Shank3 heterozygous knockout mice mostly showed minor abnormalities to full knockouts, which might reflect the importance of proper Shank3 dosage in neuronal cells. Most reported volume changes seemed to be more pronounced at younger age. Our results indicate both convergent and divergent brain region abnormalities in genetic and non-genetic models of ASD. These alterations of brain structures might be mirrored in the reported behavior of both models, which have not been assessed in this study.

Sex-dependent neurobiological features of prenatal immune activation via TLR7

Immune activation during pregnancy via infection or autoimmune disease is a risk factor for neuropsychiatric illness. Mouse models of prenatal immune activation often involve maternal administration of agents that activate toll-like receptors (TLRs), a class of pattern recognition receptors that initiate innate immune responses. Such studies have focused primarily on activating the TLR3 or TLR4 subtypes, to mimic immune responses to viral or bacterial infections, respectively. Here, we characterize the effects of prenatal activation of TLR7, which is implicated in the pathogenesis of autoimmune disease. Prenatal TLR7 activation via administration of the selective agonist imiquimod (5.0 mg/kg) induces a phenotype in offspring characterized by reduced anxiety-like behavior, fragmented social behavior, and altered ultrasonic vocalization patterns at 6–12 weeks of age. The characteristics of this phenotype are readily distinguishable from—and in some ways opposite to—those seen following prenatal activation of TLR3 and/or TLR4. Prenatal TLR7-activated mice have normal baseline locomotor activity, but are hyperresponsive to stimuli including social partners, circadian cues, and gonadal hormone fluctuations. These alterations are accompanied by decreases in microglia density but increases in ramifications. RNA-sequencing of dorsal striatum, a region showing profound changes in microglial markers, indicates that prenatal TLR7 activation induces differential expression of hundreds of genes at 13 weeks of age, with virtually no overlap in differentially expressed genes between males and females. Our findings demonstrate that prenatal immune activation can promote a wide range of developmental trajectories, depending on the type and/or pattern of TLR activation and the sex of the offspring.

Prenatal growth map of the mouse knee joint by means of deformable registration technique.

Joint morphogenesis is the process during which distinct and functional joint shapes emerge during pre- and post-natal joint development. In this study, a repeatable semi-automatic protocol capable of providing a 3D realistic developmental map of the prenatal mouse knee joint was designed by combining Optical Projection Tomography imaging (OPT) and a deformable registration algorithm (Sheffield Image Registration toolkit, ShIRT). Eleven left limbs of healthy murine embryos were scanned with OPT (voxel size: 14.63μm) at two different stages of development: Theiler stage (TS) 23 (approximately 14.5 embryonic days) and 24 (approximately 15.5 embryonic days). One TS23 limb was used to evaluate the precision of the displacement predictions for this specific case. The remaining limbs were then used to estimate Developmental Tibia and Femur Maps. Acceptable uncertainties of the displacement predictions computed from repeated images were found for both epiphyses (between 1.3μm and 1.4μm for the proximal tibia and between 0.7μm and 1.0μm for the femur, along all directions). The protocol was found to be reproducible with maximum Modified Housdorff Distance (MHD) differences equal to 1.9 μm and 1.5 μm for the tibial and femoral epiphyses respectively. The effect of the initial shape of the rudiment affected the developmental maps with MHD of 21.7 μm and 21.9 μm for the tibial and femoral epiphyses respectively, which correspond to 1.4 and 1.5 times the voxel size. To conclude, this study proposes a repeatable semi-automatic protocol capable of providing mean 3D realistic developmental map of a developing rudiment allowing researchers to study how growth and adaptation are directed by biological and mechanobiological factors.

Epigenetic Alterations in Prenatal Stress Mice as an Endophenotype Model for Schizophrenia: Role of Metabotropic Glutamate 2/3 Receptors.

Mice subjected to prenatal restraint stress (PRS mice) showed biochemical and behavioral abnormalities consistent with a schizophrenia-like phenotype (Matrisciano et al., 2016). PRS mice are characterized by increased DNA-methyltransferase 1 (DNMT1) and ten-eleven methylcytosine dioxygenase 1 (TET1) expression levels and exhibit an enrichment of 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) at neocortical GABAergic and glutamatergic gene promoters. Activation of group II metabotropic glutamate receptors (mGlu2 and−3 receptors) showed a potential epigenetically-induced antipsychotic activity by reversing the molecular and behavioral changes observed in PRS mice. This effect was most likely caused by the increase in the expression of growth arrest and DNA damage 45-β (Gadd45-β) protein, a molecular player of DNA demethylation, induced by the activation of mGlu2/3 receptors. This effect was mimicked by clozapine and valproate but not by haloperidol. Treatment with the selective mGlu2/3 receptors agonist LY379268 also increased the amount of Gadd45-β bound to specific promoter regions of reelin, BDNF, and GAD67. A meta-analysis of several clinical trials showed that treatment with an orthosteric mGlu2/3 receptor agonist improved both positive and negative symptoms of schizophrenia, but only in patients who were early-in-disease and had not been treated with atypical antipsychotic drugs (Kinon et al., 2015). Our findings show that PRS mice are valuable model for the study of epigenetic mechanisms involved in the pathogenesis of schizophrenia and support the hypothesis that pharmacological modulation of mGlu2/3 receptors could impact the early phase of schizophrenia and related neurodevelopmental disorders by regulating epigenetic processes that lie at the core of the disorders.

MS-11, a mimetic of the mSin3-binding helix in NRSF, ameliorates social interaction deficits in a prenatal valproic acid-induced autism mouse model

Growing evidence suggests pivotal roles for epigenetic mechanisms in both animal models of and individuals with autism spectrum disorders (ASD). Neuron-restrictive silencer factor (NRSF) binds to neuron-restrictive silencing elements in neuronal genes and recruits co-repressors, such as mSin3, to epigenetically inhibit neuronal gene expression. Because dysregulation of NRSF is related to ASD, here we examined the effects of mS-11, a chemically optimized mimetic of the mSin3-binding helix in NRSF, on the behavioral and morphological abnormalities found in a mouse model of valproic acid (VPA)-induced ASD. Chronic treatment with mS-11 improved prenatal VPA-induced deficits in social interaction. Additionally, we found that NRSF mRNA expression was greater in the somatosensory cortex of VPA-exposed mice than of controls. Agreeing with these behavioral findings, mice that were prenatally exposed to VPA showed lower dendritic spine density in the somatosensory cortex, which was reversed by chronic treatment with mS-11. These findings suggest that mS-11 has the potential for improving ASD-related symptoms through inhibition of mSin3-NRSF binding.

Sex-specific deficits in biochemical but not behavioral responses to delay fear conditioning in prenatal alcohol exposure mice.

Studies in clinical populations and preclinical models have shown that prenatal alcohol exposure (PAE) is associated with impairments in the acquisition, consolidation and recall of information, with deficits in hippocampal formation-dependent learning and memory being a common finding. The glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and extracellular signal-regulated kinase 2 (ERK2) are key regulators of hippocampal formation development, structure and functioning and, thus, are potential mediators of PAE's effects on this brain region. In the present studies, we employed a well-characterized mouse model of PAE to identify biochemical mechanisms that may underlie activity-dependent learning and memory deficits associated with PAE. Mouse dams consumed either 10% (w/v) ethanol in 0.066% (w/v) saccharin (SAC) or 0.066% (w/v) SAC alone using a limited (4-h) access, drinking-in-the-dark paradigm. Male and female offspring (∼180-days of age) were trained using a delay conditioning procedure and contextual fear responses (freezing behavior) were measured 24 h later. Hippocampal formation tissue and blood were collected from three behavioral groups of animals: 20 min following conditioning (conditioning only group), 20 min following the re-exposure to the context (conditioning plus re-exposure group), and behaviorally naïve (naïve group) mice. Plasma corticosterone levels were measured by enzyme immunoassay. Immunoblotting techniques were used to measure protein levels of the GR, MR, ERK1 and ERK2 in nuclear and membrane fractions prepared from the hippocampal formation. Adult SAC control male and female mice displayed similar levels of contextual fear. However, significant sex differences were observed in freezing exhibited during the conditioning session. Compared to same-sex SAC controls, male and female PAE mice demonstrated context fear deficits While plasma corticosterone concentrations were elevated in PAE males and females relative to their respective SAC naïve controls, plasma corticosterone concentrations in the conditioning only and conditioning plus re-exposure groups were similar in SAC and PAE animals. Relative to the respective naïve group, nuclear GR protein levels were increased in SAC, but not PAE, male hippocampal formation in the conditioning only group. In contrast, no difference was observed between nuclear GR levels in the naïve and conditioning plus re-exposure groups. In females, nuclear GR levels were significantly reduced by PAE but there was no effect of behavioral group or interaction between prenatal treatment and behavioral group. In males, nuclear MR levels were significantly elevated in the SAC conditioning plus re-exposure group compared to SAC naïve mice. In PAE females, nuclear MR levels were elevated in both the conditioning only and conditioning plus re-exposure groups relative to the naïve group. Levels of activated ERK2 (phospho-ERK2 expressed relative to total ERK2) protein were elevated in SAC, but not PAE, males following context re-exposure, and a significant interaction between prenatal exposure group and behavioral group was found. No main effects or interactions of behavioral group and prenatal treatment on nuclear ERK2 were found in female mice. These findings suggest a sex difference in which molecular pathways are activated during fear conditioning in mice. In PAE males, the deficits in contextual fear were associated with the loss of responsiveness of hippocampal formation nuclear GR, MR and ERK2 to signals generated by fear conditioning and context re-exposure. In contrast, the contextual fear deficit in PAE female mice does not appear to be associated with activity-dependent changes in GR and MR levels or ERK2 activation during training or memory recall, although an overall reduction in nuclear GR levels may play a role. These studies add to a growing body of literature demonstrating that, at least partially, different mechanisms underlie learning, memory formation and memory recall in males and females and that these pathways are differentially affected by PAE.

The histamine H3R antagonist DL77 attenuates autistic behaviors in a prenatal valproic acid-induced mouse model of autism

Autistic spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairment in social communication and restricted/repetitive behavior patterns or interests. Antagonists targeting histamine H3 receptor (H3R) are considered potential therapeutic agents for the therapeutic management of different brain disorders, e.g., cognitive impairments. Therefore, the effects of subchronic treatment with the potent and selective H3R antagonist DL77 (5, 10, or 15 mg/kg, i.p.) on sociability, social novelty, anxiety, and aggressive/repetitive behavior in male Tuck-Ordinary (TO) mice with ASD-like behaviors induced by prenatal exposure to valproic acid (VPA, 500 mg/kg, i.p.) were evaluated using the three-chamber test (TCT), marble burying test (MBT), nestlet shredding test (NST), and elevated plus maze (EPM) test. The results showed that VPA-exposed mice exhibited significantly lower sociability and social novelty preference compared to VPA-exposed mice that were pretreated with DL77 (10 or 15 mg/kg, i.p.). VPA-exposed mice presented a significantly higher percentage of buried marbles in MBT and shredded nestlet significantly more in NST compared to the control groups. However, VPA-exposed animals pretreated with DL77 (10 or 15 mg/kg, i.p.) buried a reduced percentage of marbles in MBT and presented a significantly lower percentage of shredding behavior in NST. On the other hand, pretreatment with DL77 (5, 10, or 15 mg/kg, i.p.) failed to restore the disturbed anxiety levels and hyperactivity observed in VPA-exposed animals in EPM, whereas the reference drug donepezil (DOZ, 1 mg/kg, i.p.) significantly palliated the anxiety and reduced the hyperactivity measures of VPA-exposed mice. Furthermore, pretreatment with DL77 (10 or 15 mg/kg, i.p.) modulated oxidative stress status by increasing GSH and decreasing MDA, and it attenuated the proinflammatory cytokines IL-1β, IL-6 and TNF-α exacerbated by lipopolysaccharide (LPS) challenge, in VPA-exposed mouse brain tissue. Taken together, these results provide evidence that modulation of brain histaminergic neurotransmission, such as by subchronic administration of the H3R antagonist DL77, may serve as an effective pharmacological therapeutic target to rescue ASD-like behaviors in VPA-exposed animals, although further investigations are necessary to corroborate and expand these initial data.

How to choose the suitable animal model of polycystic ovary syndrome?

Polycystic ovary syndrome (PCOS) is a gynecological metabolic and endocrine disorder with uncertain etiology. To understand the etiology of PCOS or the evaluation of various therapeutic agents, different animal models have been introduced. Considering this fact that is difficult to develop an animal model that mimics all aspects of this syndrome, but, similarity of biological, anatomical, and/or biochemical features of animal model to the human PCOS phenotypes can increase its application. This review paper evaluates the recently researched animal models and introduced the best models for different research purposes in PCOS studies. During January 2013 to January 2017, 162 studies were identified which applied various kinds of animal models of PCOS including rodent, primate, ruminant and fish. Between these models, prenatal and pre-pubertal androgen rat models and then prenatal androgen mouse model have been studied in detail than others. The comparison of main features of these models with women PCOS demonstrates higher similarity of these three models to human conditions. Thereafter, letrozole models can be recommended for the investigation of various aspects of PCOS. Interestingly, similarity of PCOS features of post-pubertal insulin and human chorionic gonadotropin rat models with women PCOS were considerable which can make it as a good choice for future investigations.