Dana L. Shuey

Serotonin uptake in the ectoplacental cone and placenta of the mouse

The neurotransmitter serotonin (5-HT) was localized in the ectoplacental cone (EPC) and placenta of the day 9-12 (E9-12) mouse embryo in vivo and in whole embryo cultures, using immunocytochemistry with a specific 5-HT antiserum. In uncultured conceptuses, 5-HT immunoreactivity (5-HT IR) was most intense in the EPC at E9 (2-7 somites), particularly in giant cells around the periphery. Nuclear staining was observed in lightly staining giant cells and in small cells in the core of the cone. By E10 (18-24 somites) 5-HT IR in the placenta was less intense and almost exclusively limited to giant cells, where it was localized to chromatin-like material in nuclei. The same pattern and level of 5-HT IR persisted through E12. In the placenta, 5-HT IR appeared to be most intense in giant cells located near aggregations of platelets in decidual blood vessels. 5-HT IR was enhanced in cultured conceptuses, and further increased when exogenous 5-HT was added to the culture medium. Immunoreactivity was greatly reduced by adding the 5-HT uptake inhibitor fluoxetine to the culture medium, or culturing conceptuses in medium containing 5-HT depleted rat serum. Thus, 5-HT was apparently taken up from the culture medium. In conceptuses exposed to exogenous 5-HT, immunoreactivity in the placenta appeared as a gradient from the giant cells to the inner layers, suggesting that these cells may transport 5-HT toward the embryo. No evidence of 5-HT synthesis by the EPC/placenta was found. These results suggest that 5-HT present in the EPC/placenta is due to uptake, not synthesis. Possible sources and functions of 5-HT in the developing placenta are discussed.

Serotonin and morphogenesis

SummaryThis study describes the timecourse of expression of low-affinity serotonin uptake sites in the developing craniofacial region of the mouse embryo. Whole mouse embryos were incubated in the presence of various serotonergic compounds followed by immunocyto-chemical localization of serotonin (5-HT) and its binding protein. In the gestational day 9 embryo (3–5 somites), 5-HT uptake was observed in the myocardium of the heart, the visceral yolk sac and foregut. A specific and transient pattern of 5-HT uptake was observed in the hindbrain neuroepithelium from day 9.5–11, where it was localized in rhombomeres 2–5 in the day 9.5 embryo. By day 10, when rhombomeres were no longer evident, uptake was present in the dorso-lateral neuroepithelium surrounding the fourth ventricle (rhombic lip; cerebellar anlage). Uptake of 5-HT was initially observed in the surface epithelium of the craniofacial region at day 10 (20–25 somites) and was greatly increased at day 11. The invaginating lens, nasal placode epithelium and otocyst also took up 5-HT at day 11. During these stages a 45 kD serotonin-binding protein (SBP) was expressed in craniofacial mesenchyme, and became progressively restricted to regions subjacent to epithelial uptake sites. These staining patterns were shown to be specific for 5-HT and SBP by their absence in embryos stained using preabsorbed antisera. The timecourse of these patterns are correlated with critical events in craniofacial morphogenesis including (1) onset of inductive epithelial-mesenchymal interactions, (2) invagination and fusion of placodal structures, (3) presence of rhombomeres, and (4) regions of low proliferative activity.

Biological modeling of 5-fluorouracil developmental toxicity

A biologically-based dose-response (BBDR) model is a mathematical description of the biological events leading to expression of a toxic response. As an alternative to current approaches in non-cancer risk assessment, such models will reduce uncertainty in that they will provide a more comprehensive description of toxicity. We are involved in construction of a BBDR model for the developmental toxicity of 5-fluorouracil (5-FU) in the rat using multiple approaches. First, to identify critical events in the pathogenesis of 5-FU developmental toxicity, thymidylate synthetase (TS) inhibition and alterations in cell kinetics and growth were examined in embryos following maternal administration of 5-FU on day 14 of gestation. A dose-related decline in TS activity was observed within 1 h; however, maximal inhibition and recovery were similar at 10, 20 and 40 mg/kg. Dose-dependent cell cycle alterations were observed within 4 h after exposure and were maximal at 8 h. Hindlimb growth reduction was observed 24 h after exposure to 40 mg/kg, but not at lower doses. At term hindlimb defects were observed at doses above 30 mg/kg. An integrated dose-response model for hindlimb defects was derived from empirical relationships among these events. The resultant dose-response somewhat over-predicted the developmental toxicity of 5-FU, although results of a Monte Carlo simulation indicated that these data were not incompatible with model predictions. Overall, the results suggest that TS inhibition is a key component of the mechanism of 5-FU developmental toxicology, but the model does not capture all of the critical events in the induction of hindlimb defects. A preliminary mechanistic model for the inhibition of embryonic TS, DNA synthesis and cell cycle following maternal exposure to 5-FU, independently derived from literature data to further examine the potential role of this pathway in its developmental toxicity, predicted a dose-response for TS inhibition and DNA synthesis that closely reflected the observed patterns. These results further suggest that TS inhibition, resultant deficits in DNA synthesis and cell cycle perturbations represent a critical mechanistic pathway in the developmental toxicity of 5-FU.

Fetal hippocampal cell suspensions ameliorate behavioral effects of intradentate colchicine in the rat

Colchicine, a neurotoxin that preferentially destroys dentate gyrus granule cells and mossy fibers, was injected into the hippocampus of adult rats. Three weeks later, the rats were tested for colchicine-induced hypermotility after which they received fetal hippocampal explants. Locomotor activity was retested three weeks later, after which the rats were trained over a period of four weeks on a food-reinforced, spatial, working memory task in an 8-arm radial maze. Fetal hippocampal explants were found to attenuate significantly the colchicine-induced hypermotility and spatial learning deficits. Histological observations showed the presence of surviving hippocampal explants in both the lesioned and the control rat brains, suggesting that the presence of viable implants facilitates the recovery of behavioral function in rats with spatial memory deficits.