Developmental neurotoxicity of environmentally relevant pharmaceuticals and mixtures thereof in a zebrafish embryo behavioural test

Humans are exposed daily to complex mixtures of chemical substances via food intake, inhalation, and dermal contact. Developmental neurotoxicity is an understudied area and entails one of the most complex areas in toxicology. Animal studies for developmental neurotoxicity (DNT) are hardly performed in the context of regular hazard studies, as they are costly and time consuming and provide only limited information as to human relevance. There is a need for a combination of in vitro and in silico tests for the assessment of chemically induced DNT in humans. The zebrafish (Danio rerio) embryo (ZFE) provides a powerful model to study DNT because it shows fast neurodevelopment with a large resemblance to the higher vertebrate, including the human system. One of the suitable readouts for DNT testing in the zebrafish is neurobehaviour (stimulus-provoked locomotion) since this provides integrated information on the functionality and status of the entire nervous system of the embryo. In the current study, environmentally relevant pharmaceuticals and their mixtures were investigated using the zebrafish light-dark transition test. Zebrafish embryos were exposed to three neuroactive compounds of concern, carbamazepine (CBZ), fluoxetine (FLX), and venlafaxine (VNX), as well as their main metabolites, carbamazepine 10,11-epoxide (CBZ 10,11E), norfluoxetine (norFLX), and desvenlafaxine (desVNX). All the studied compounds, except CBZ 10,11E, dose-dependently inhibited zebrafish locomotor activity, providing a distinct behavioural phenotype. Mixture experiments with these pharmaceuticals identified that dose addition was confirmed for all the studied binary mixtures (CBZ-FLX, CBZ-VNX, and VNX-FLX), thereby supporting the zebrafish embryo as a model for studying the cumulative effect of chemical mixtures in DNT. This study shows that pharmaceuticals and a mixture thereof affect locomotor activity in zebrafish. The test is directly applicable in environmental risk assessment; however, further studies are required to assess the relevance of these findings for developmental neurotoxicity in humans

Galectin-1 as a marker for microglia activation in the aging brain

Microglia cells, the immune cells residing in the brain, express immune regulatory molecules that have a central role in the manifestation of age-related brain characteristics. Our hypothesis suggests that galectin-1, an anti-inflammatory member of the beta-galactoside-binding lectin family, regulates microglia and neuroinflammation in the aging brain. Through our in-silico analysis, we discovered a subcluster of microglia in the aged mouse brain that exhibited increased expression of galectin-1 mRNA. In our Western blotting experiments, we observed a decrease in galectin-1 protein content in our rat primary cortical cultures over time. Additionally, we found that the presence of lipopolysaccharide, an immune activator, significantly increased the expression of galectin-1 protein in microglial cells. Utilizing flow cytometry, we determined that a portion of the galectin-1 protein was localized on the surface of the microglial cells. As cultivation time increased, we observed a decrease in the expression of activation-coupled molecules in microglial cells, indicating cellular exhaustion. In our mixed rat primary cortical cell cultures, we noted a transition of amoeboid microglial cells labeled with OX42(CD11b/c) to a ramified, branched phenotype during extended cultivation, accompanied by a complete disappearance of galectin-1 expression. By analyzing the transcriptome of a distinct microglial subpopulation in an animal model of aging, we established a correlation between chronological aging and galectin-1 expression. Furthermore, our in vitro study demonstrated that galectin-1 expression is associated with the functional activation state of microglial cells exhibiting specific amoeboid morphological characteristics. Based on our findings, we identify galectin-1 as a marker for microglia activation in the context of aging

Accelerated Postnatal Growth Increases Lipogenic Gene Expression and Adipocyte Size in Low–Birth Weight Mice

OBJECTIVE: To characterize the hormonal milieu and adipose gene expression in response to catch-up growth (CUG), a growth pattern associated with obesity and diabetes risk, in a mouse model of low birth weight (LBW). RESEARCH DESIGN AND METHODS: ICR mice were food restricted by 50% from gestational days 12.5–18.5, reducing offspring birth weight by 25%. During the suckling period, dams were either fed ad libitum, permitting CUG in offspring, or food restricted, preventing CUG. Offspring were killed at age 3 weeks, and gonadal fat was removed for RNA extraction, array analysis, RT-PCR, and evaluation of cell size and number. Serum insulin, thyroxine (T4), corticosterone, and adipokines were measured. RESULTS: At age 3 weeks, LBW mice with CUG (designated U-C) had body weight comparable with controls (designated C-C); weight was reduced by 49% in LBW mice without CUG (designated U-U). Adiposity was altered by postnatal nutrition, with gonadal fat increased by 50% in U-C and decreased by 58% in U-U mice (P less than 0.05 vs. C-C mice). Adipose expression of the lipogenic genes Fasn, AccI, Lpin1, and Srebf1 was significantly increased in U-C compared with both C-C and U-U mice (P less than 0.05). Mitochondrial DNA copy number was reduced by greater than 50% in U-C versus U-U mice (P = 0.014). Although cell numbers did not differ, mean adipocyte diameter was increased in U-C and reduced in U-U mice (P less than 0.01). CONCLUSIONS: CUG results in increased adipose tissue lipogenic gene expression and adipocyte diameter but not increased cellularity, suggesting that catch-up fat is primarily associated with lipogenesis rather than adipogenesis in this murine model

Lmx1b is required for the glutamatergic fates of a subset of spinal cord neurons

Background: Alterations in neurotransmitter phenotypes of specific neurons can cause imbalances in excitation and inhibition in the central nervous system (CNS), leading to diseases. Therefore, the correct specification and maintenance of neurotransmitter phenotypes is vital. As with other neuronal properties, neurotransmitter phenotypes are often specified and maintained by particular transcription factors. However, the specific molecular mechanisms and transcription factors that regulate neurotransmitter phenotypes remain largely unknown. Methods: In this paper we use single mutant, double mutant and transgenic zebrafish embryos to elucidate the functions of Lmx1ba and Lmx1bb in the regulation of spinal cord interneuron neurotransmitter phenotypes. Results: We demonstrate that lmx1ba and lmx1bb are both expressed in zebrafish spinal cord and that lmx1bb is expressed by both V0v cells and dI5 cells. Our functional analyses demonstrate that these transcription factors are not required for neurotransmitter fate specification at early stages of development, but that in embryos with at least two lmx1ba and/or lmx1bb mutant alleles there is a reduced number of excitatory (glutamatergic) spinal interneurons at later stages of development. In contrast, there is no change in the numbers of V0v or dI5 cells. These data suggest that lmx1b-expressing spinal neurons still form normally, but at least a subset of them lose, or do not form, their normal excitatory fates. As the reduction in glutamatergic cells is only seen at later stages of development, Lmx1b is probably required either for the maintenance of glutamatergic fates or to specify glutamatergic phenotypes of a subset of later forming neurons. Using double labeling experiments, we also show that at least some of the cells that lose their normal glutamatergic phenotype are V0v cells. Finally, we also establish that Evx1 and Evx2, two transcription factors that are required for V0v cells to acquire their excitatory neurotransmitter phenotype, are also required for lmx1ba and lmx1bb expression in these cells, suggesting that Lmx1ba and Lmx1bb act downstream of Evx1 and Evx2 in V0v cells. Conclusions: Lmx1ba and Lmx1bb function at least partially redundantly in the spinal cord and three functional lmx1b alleles are required in zebrafish for correct numbers of excitatory spinal interneurons at later developmental stages. Taken together, our data significantly enhance our understanding of how spinal cord neurotransmitter fates are regulated