Sex differences in hippocampal structural plasticity and glycosaminoglycan disaccharide levels after neonatal handling.

In this study we investigated the effects of a neonatal handling protocol that mimics the handling of sham control pups in protocols of neonatal exposure to brain insults on dendritic arborization and glycosaminoglycan (GAG) levels in the developing brain. GAGs are long, unbranched polysaccharides, consisting of repeating disaccharide units that can be modified by sulfation at specific sites and are involved in modulating neuronal plasticity during brain development. In this study, male and female Sprague-Dawley rats underwent neonatal handling daily between post-natal day (PD)4 and PD9, with brains analyzed on PD9. Neuronal morphology and morphometric analysis of the apical and basal dendritic trees of CA1 hippocampal pyramidal neurons were carried out by Golgi-Cox staining followed by neuron tracing and analysis with the software Neurolucida. Chondroitin sulfate (CS)-, Hyaluronic Acid (HA)-, and Heparan Sulfate (HS)-GAG disaccharide levels were quantified in the hippocampus by Liquid Chromatography/Mass Spectrometry analyses. We found sex by neonatal handling interactions on several parameters of CA1 pyramidal neuron morphology and in the levels of HS-GAGs, with females, but not males, showing an increase in both dendritic arborization and HS-GAG levels. We also observed increased expression of glucocorticoid receptor gene Nr3c1 in the hippocampus of both males and females following neonatal handling suggesting that both sexes experienced a similar stress during the handling procedure. This is the first study to show sex differences in two parameters of brain plasticity, CA1 neuron morphology and HS-GAG levels, following handling stress in neonatal rats.

The potential involvement of inhaled iron (Fe) in the neurotoxic effects of ultrafine particulate matter air pollution exposure on brain development in mice.

BACKGROUND: Air pollution has been associated with neurodevelopmental disorders in epidemiological studies. In our studies in mice, developmental exposures to ambient ultrafine particulate (UFP) matter either postnatally or gestationally results in neurotoxic consequences that include brain metal dyshomeostasis, including significant increases in brain Fe. Since Fe is redox active and neurotoxic to brain in excess, this study examined the extent to which postnatal Fe inhalation exposure, might contribute to the observed neurotoxicity of UFPs. Mice were exposed to 1 µg/m3 Fe oxide nanoparticles alone, or in conjunction with sulfur dioxide (Fe (1 µg/m3) + SO2 (SO2 at 1.31 mg/m3, 500 ppb) from postnatal days 4-7 and 10-13 for 4 h/day. RESULTS: Overarching results included the observations that Fe + SO2 produced greater neurotoxicity than did Fe alone, that females appeared to show greater vulnerability to these exposures than did males, and that profiles of effects differed by sex. Consistent with metal dyshomeostasis, both Fe only and Fe + SO2 exposures altered correlations of Fe and of sulfur (S) with other metals in a sex and tissue-specific manner. Specifically, altered metal levels in lung, but particularly in frontal cortex were found, with reductions produced by Fe in females, but increases produced by Fe + SO2 in males. At PND14, marked changes in brain frontal cortex and striatal neurotransmitter systems were observed, particularly in response to combined Fe + SO2 as compared to Fe only, in glutamatergic and dopaminergic functions that were of opposite directions by sex. Changes in markers of trans-sulfuration in frontal cortex likewise differed in females as compared to males. Residual neurotransmitter changes were limited at PND60. Increases in serum glutathione and Il-1a were female-specific effects of combined Fe + SO2. CONCLUSIONS: Collectively, these findings suggest a role for the Fe contamination in air pollution in the observed neurotoxicity of ambient UFPs and that such involvement may be different by chemical mixture. Translation of such results to humans requires verification, and, if found, would suggest a need for regulation of Fe in air for public health protection.

Evaluating the Effects of BPA and TBBPA Exposure on Pregnancy Loss and Maternal-Fetal Immune Cells in Mice.

BACKGROUND: Bisphenol A (BPA) exposure has been linked to miscarriages and pregnancy complications in humans. In contrast, the potential reproductive toxicity of BPA analogs, including tetrabromobisphenol A (TBBPA), is understudied. Furthermore, although environmental exposure has been linked to altered immune mediators, the effects of BPA and TBBPA on maternal-fetal immune tolerance during pregnancy have not been studied. The present study investigated whether exposure resulted in higher rates of pregnancy loss in mice, lower number of regulatory T cells (Tregs), and lower indoleamine 2,3 deoxygenase 1 (Ido1) expression, which provided evidence for mechanisms related to immune tolerance in pregnancy. OBJECTIVES: The purpose of this investigation was to characterize the effects of BPA and TBBPA exposure on pregnancy loss in mice and to study the percentage and number of Tregs and Ido1 expression and DNA methylation. METHODS: Analysis of fetal resorption and quantification of maternal and fetal immune cells by flow cytometry were performed in allogeneic and syngeneic pregnancies. Ido1 mRNA and protein expression, and DNA methylation in placentas from control and BPA- and TBBPA-exposed mice were analyzed using real-time quantitative polymerase chain reaction, immunofluorescence, and bisulfite sequencing analyses. RESULTS: BPA and TBBPA exposure resulted in higher rates of hemorrhaging in early allogeneic, but not syngeneic, conceptuses. In allogeneic pregnancies, BPA and TBBPA exposure was associated with higher fetal resorption rates and lower maternal Treg number. Importantly, these differences were associated with lower IDO1 protein expression in trophoblast giant cells and higher mean percentage Ido1 DNA methylation in embryonic day 9.5 placentas from BPA- and TBBPA-exposed mice. DISCUSSION: BPA- and TBBPA-induced pregnancy loss in mice was associated with perturbed IDO1-dependent maternal immune tolerance. https://doi.org/10.1289/EHP10640.

Astrocyte tissue plasminogen activator expression during brain development and its role in pyramidal neuron neurite outgrowth.

The serine protease tissue plasminogen activator (tPA), encoded by the gene Plat, exerts a wide range of proteolysis-dependent and proteolysis-independent functions. In the developing brain, tPA is involved in neuronal development via the modulation of the proteolytic degradation of the extracellular matrix (ECM). Both lack of and excessive tPA are associated with neurodevelopmental disorders and with brain pathology. Astrocytes play a major role in neurite outgrowth of developing neurons as they are major producers of ECM proteins and ECM proteases. In this study we investigated the expression of Plat in developing and mature hippocampal and cortical astrocytes of Aldh1l1-EGFP-Rpl10a mice in vivo following Translating Ribosome Affinity Purification (TRAP) and the role of tPA in modulating astrocyte-mediated neurite outgrowth in an in vitro astrocyte-neuron co-culture system. We show that Plat is highly enriched in astrocytes in the developing, but not in the mature, hippocampus and cortex. Both the silencing of tPA expression in astrocytes and astrocyte exposure to recombinant tPA reduce neuritogenesis in co-cultured hippocampal neurons. These results suggest that astrocyte tPA is involved in modulating neuronal development and that tight control of astrocyte tPA expression is important for normal neuronal development, with both experimentally elevated and reduced levels of this proteolytic enzyme impairing neurite outgrowth. These results are consistent with the hypothesis that the ECM, by serving as adhesive substrate, enables neurite outgrowth, but that controlled proteolysis of the ECM is needed for growth cone advancement.

Effects of ethanol-and choline-treated astrocytes on hippocampal neuron neurite outgrowth in vitro.

Exposure to ethanol in utero can result in Fetal Alcohol Spectrum Disorders, which may cause long-lasting cognitive and behavioral abnormalities. Preclinical studies indicate that choline ameliorates the behavioral effects of developmental alcohol exposure in rodents, and clinical studies on the effectiveness of choline, administered early in pregnancy, showed that the adverse effects of heavy prenatal alcohol exposure on postnatal growth, and cognition in human infants were mitigated. However, little is known on the mechanisms behind the effects of choline. We have previously reported that astrocyte pre-treatment with 75 mM ethanol, in vitro, reduces neurite outgrowth in hippocampal neurons co-cultured with the pre-treated astrocytes. Our in vitro system allows us to study the effects of chemicals on astrocyte functions, able to modulate neuronal development. The main objective was to test the hypothesis that choline can ameliorate the astrocyte-mediated effects of ethanol on neurite growth. In this study, we exposed primary rat cortical astrocytes to ethanol, choline, ethanol plus choline, or control conditions for 24 h. Culture media was then removed, replaced with fresh media containing no ethanol or choline treatments and primary rat hippocampal neurons were plated on top of the astrocyte monolayer and cultured for 16 h. Neurons were then stained for ß-III Tubulin and neurite outgrowth was measured. Astrocyte exposure to ethanol (25, 50, and 75 mM) decreases neurite outgrowth in co-cultured hippocampal pyramidal neurons, while astrocyte treatment with choline had no effect. Astrocyte treatment with ethanol and choline in combination, however, prevented the effect of ethanol, leading to levels of neurite outgrowth similar the control condition. Choline prevents the inhibitory effect of ethanol-treated astrocytes on neurite outgrowth while not altering normal neuronal development. These results suggest a new, astrocyte-mediated mechanism by which choline ameliorates the effects of developmental alcohol exposure.

Chronic Chemogenetic Stimulation of the Nucleus Accumbens Produces Lasting Reductions in Binge Drinking and Ameliorates Alcohol-Related Morphological and Transcriptional Changes.

Binge drinking is a dangerous pattern of behavior. We tested whether chronically manipulating nucleus accumbens (NAc) activity (via clozapine-N-oxide (CNO) and Designer Receptors Exclusively Activated by Designer Drugs (DREADD)) could produce lasting effects on ethanol binge-like drinking in mice selectively bred to drink to intoxication. We found chronically increasing NAc activity (4 weeks, via CNO and the excitatory DREADD, hM3Dq) decreased binge-like drinking, but did not observe CNO-induced changes in drinking with the inhibitory DREADD, hM4Di. The CNO/hM3Dq-induced reduction in ethanol drinking persisted for at least one week, suggesting adaptive neuroplasticity via transcriptional and epigenetic mechanisms. Therefore, we defined this plasticity at the morphological and transcriptomic levels. We found that chronic binge drinking (6 weeks) altered neuronal morphology in the NAc, an effect that was ameliorated with CNO/hM3Dq. Moreover, we detected significant changes in expression of several plasticity-related genes with binge drinking that were ameliorated with CNO treatment (e.g., Hdac4). Lastly, we found that LMK235, an HDAC4/5 inhibitor, reduced binge-like drinking. Thus, we were able to target specific molecular pathways using pharmacology to mimic the behavioral effects of DREADDs.

Plasminogen activator system homeostasis and its dysregulation by ethanol in astrocyte cultures and the developing brain.

In utero alcohol exposure can cause fetal alcohol spectrum disorders (FASD), characterized by structural brain abnormalities and long-lasting behavioral and cognitive dysfunction. Neuronal plasticity is affected by in utero alcohol exposure and can be modulated by extracellular proteolysis. Plasmin is a major extracellular serine-protease whose activation is tightly regulated by the plasminogen activator (PA) system. In the present study we explored the effect of ethanol on the expression of the main components of the brain PA system in sex-specific cortical astrocyte primary cultures in vitro and in the cortex and hippocampus of post-natal day (PD) 9 male and female rats. We find that ethanol alters the PA system in astrocytes and in the developing brain. In particular, the expression of tissue-type PA (tPA), encoded by the gene Plat, is consistently upregulated by ethanol in astrocytes in vitro and in the cortex and hippocampus in vivo. Astrocytes exhibit endogenous plasmin activity that is increased by ethanol and recombinant tPA and inhibited by tPA silencing. We also find that tPA is expressed by astrocytes of the developing cortex and hippocampus in vivo. All components of the PA system investigated, with the exception of Neuroserpin/Serpini1, are expressed at higher levels in astrocyte cultures than in the developing brain, suggesting that astrocytes are major producers of these proteins in the brain. In conclusion, astrocyte PA system may play a major role in the modulation of neuronal plasticity; ethanol-induced upregulation of tPA levels and plasmin activity may be responsible for altered neuronal plasticity in FASD.