Impaired dendritic growth and positioning of cortical pyramidal neurons by activation of aryl hydrocarbon receptor signaling in the developing mouse.

The basic helix-loop-helix (bHLH) transcription factors exert multiple functions in mammalian cerebral cortex development. The aryl hydrocarbon receptor (AhR), a member of the bHLH-Per-Arnt-Sim subfamily, is a ligand-activated transcription factor reported to regulate nervous system development in both invertebrates and vertebrates, but the functions that AhR signaling pathway may have for mammalian cerebral cortex development remains elusive. Although the endogenous ligand involved in brain developmental process has not been identified, the environmental pollutant dioxin potently binds AhR and induces abnormalities in higher brain function of laboratory animals. Thus, we studied how activation of AhR signaling influences cortical development in mice. To this end, we produced mice expressing either constitutively active-AhR (CA-AhR), which has the capacity for ligand-independent activation of downstream genes, or AhR, which requires its ligands for activation. In brief, CA-AhR-expressing plasmid and AhR-expressing plasmid were each transfected into neural stems cells in the developing cerebrum by in utero electroporation on embryonic day 14.5. On postnatal day 14, mice transfected in utero with CA-AhR, but not those transfected with AhR, exhibited drastically reduced dendritic arborization of layer II/III pyramidal neurons and impaired neuronal positioning in the developing somatosensory cortex. The effects of CA-AhR were observed for dendrite development but not for the commissural fiber projection, suggesting a preferential influence on dendrites. The present results indicate that over-activation of AhR perturbs neuronal migration and morphological development in mammalian cortex, supporting previous observations of impaired dendritic structure, cortical dysgenesis, and behavioral abnormalities following perinatal dioxin exposure.

In Utero Bisphenol A Exposure Induces Abnormal Neuronal Migration in the Cerebral Cortex of Mice.

Bisphenol A (BPA) has been known to have endocrine-disrupting activity to induce reproductive and behavioral abnormalities in offspring of laboratory animal species. However, morphological basis of this abnormality during brain development is largely unknown. Cerebral cortex plays a crucial role in higher brain function, and its precisely laminated structure is formed by neuronal migration. In the present study, transfecting a plasmid (pCAG-mCherry) by in utero electroporation (IUE), we visualized developing neurons and investigated the possible effects of in utero BPA exposure on neuronal migration. Pregnant mice were exposed to BPA by osmotic pump at estimated daily doses of 0, 40 (BPA-40), or 400 (BPA-400) µg/kg from embryonic day 14.5 (E14.5) to E18.5. IUE was performed at E14.5 and neuronal migration was analyzed at E18.5. Compared with the control group, neuronal migration in the cortical plate was significantly decreased in the BPA-40 group; however, there was no significant difference in the BPA-400 group. Among several neuronal migration-related genes and cortical layer-specific genes, TrkB in the BPA-400 group was found significantly upregulated. In conclusion, in utero exposure to low BPA dose was found to disrupt neuronal migration in the cerebral cortex in a dose-specific manner.

Developmental origin of abnormal dendritic growth in the mouse brain induced by in utero disruption of aryl hydrocarbon receptor signaling.

Increased prevalence of mental disorders cannot be solely attributed to genetic factors and is considered at least partly attributable to chemical exposure. Among various environmental chemicals, in utero and lactational dioxin exposure has been extensively studied and is known to induce higher brain function abnormalities in both humans and laboratory animals. However, how the perinatal dioxin exposure affects neuromorphological alterations has remained largely unknown. Therefore, in this study, we initially studied whether and how the over-expression of aryl hydrocarbon receptor (AhR), a dioxin receptor, would affect the dendritic growth in the hippocampus of the developing brain. Transfecting a constitutively active AhR plasmid into the hippocampus via in utero electroporation on gestational day (GD) 14 induced abnormal dendritic branch growth. Further, we observed that 14-day-old mice born to dams administered with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dose: 0, 0.6, or 3.0 µg/kg) on GD 12.5 exhibited disrupted dendritic branch growth in both the hippocampus and amygdala. Finally, we observed that 16-month-old mice born to dams exposed to perinatal TCDD as described above exhibited significantly reduced spine densities. These results indicated that abnormal micromorphology observed in the developing brain may persist until adulthood and may induce abnormal higher brain function later in life.

Simultaneous determination of 20 beta-agonists in pig muscle and liver by high-performance liquid chromatography/tandem mass spectrometry.

A method was developed to determine 20 illegal residual beta-agonists in pork tissues, including muscle and liver simultaneously. The samples were hydrolyzed by beta-glucuronidase, purified by PCX SPE cartridges, and detected by HPLC coupled with electrospray ionization MS/MS operating in the positive ion mode. Matrix-fortified calibration was performed to compensate for the matrix effect and loss in sample preparation. Decision limit ranged from 0.05 to 0.23 microg/kg in muscle and 0.05 to 0.57 microg/kg in liver. Decision capacity ranged from 0.11 to 0.4 microg/kg in muscle and 0.16 to 0.79 microg/kg in liver. In Food Analysis Performance Assessment Scheme proficiency test 0287, a pig liver test material containing 13 beta-agonists was analyzed using the method developed, and clenbuterol and ractopamine were confirmed as being present. Z-scores for clenbuterol and ractopamine were 0.2 and 0.6, respectively.