Interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) are cytokines with pleiotropic effects in the central nervous system (CNS), including an emerging role in neurodevelopment. This study measured the effects of cytokines on the survival of tyrosine hydroxylase (TH) immunoreactive dopamine neurons from the substantia nigra (SN), and 5-hydroxytryptamine (5-HT) immunoreactive serotonin neurons from the rostral raphe (RR), using cultures from embryonic day 14 (E14) rat brain. IL-1beta, IL-6, and TNF-alpha were added to cell cultures at 1, 10 and 100 U/ml. After 3 days in vitro, TH and 5-HT neurons were counted. The survival of 5-HT neurons was significantly reduced by 20-30% at 10 U/ml of IL-6. IL-1beta and TNF-alpha at doses of 1 and 10 U/ml appeared to have a similar effect on the survival of these neurons, but this effect was not statistically significant. Comparable non-significant reductions of survival also occurred for TH neurons at the lower doses of IL-6 and TNF-alpha. In separate experiments, SN and RR cultures were exposed to the cytokines at a higher dose (1000 U/ml), causing a significant 30-40% decrease in the survival of TH neurons, but little or no change in 5-HT neuronal survival. Taken together, these results show that IL-1beta, IL-6, and TNF-alpha can affect developing monoamine neurons at physiologically relevant concentrations, and that high doses differentially inhibit the survival of TH and 5-HT neurons after short exposures.

Expression patterns of 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors during mouse embryogenesis were investigated using highly specific monoclonal antibodies. Differential and overlapping spatio-temporal patterns of 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptor immunoreactivity were observed during active phases of morphogenesis of a variety of embryonic tissues, including neuroepithelia of brain and spinal cord, notochord, somites, cranial neural crest, craniofacial mesenchyme and epithelia, heart myocardium and endocardial cushions, tooth germs, whisker follicles, cartilage and striated muscle. The functional significance of these receptors was tested by exposing headfold stage mouse embryos to different subtype-selective 5-HT(2) receptor antagonists for 2 days in whole embryo culture. The most potent was the pan 5-HT(2) receptor antagonist ritanserin, which has high affinity for the 5-HT(2B) receptor. Ritanserin caused 100% malformed embryos at a dose of 1 microM. The 5-HT(2A/2C) receptor antagonist mianserin also caused a significant number of malformed embryos, but only when used at a 10 fold higher dose (10 microM). Ketanserin, which primarily targets 5-HT(2A) receptors, did not cause a significant number of malformed embryos at any dose tested. Together with previous evidence that 5-HT acts as an important morphoregulatory signal during mouse embryogenesis, present evidence for the early and continued expression of functional 5-HT(2) receptors throughout gestation raises the possibility that psychotropic drugs taken during pregnancy could interfere with developmental actions of 5-HT during prenatal development of neural and non-neural tissues.

Rhombencephala from rat embryos were processed as whole-mounts for immunocytochemical detection of monoaminergic cell populations, using antibodies to tyrosine hydroxylase (TH) and serotonin (5-HT). Specific advantages of the whole-mount technique over the classical serial-section method were that even isolated immunoreactive (IR) cells could be detected easily, and three-dimensional relationships could be ascertained without the need for serial reconstruction. Embryos between embryonic days (E) 12 and 16 (the day following nocturnal mating being considered as E1) were used in this study. Both TH and 5-HT immunoreactivities were already detectable at E12, even in the smallest embryos (crown-rump length: 6 mm), but there was a striking difference in the number and regional distribution of these two types of IR cells. TH was expressed in several cell groups located in the rostral rhombencephalon (the presumed anlage of the A4-7 complex) as well as in the caudal rhombencephalon (the presumed anlagen of groups A1-2 and C1-3), whereas 5-HT was expressed in very few cells located near the rostral border of the rhombencephalon (presumed anlage of the B4-9 complex). Although the three-dimensional distribution of the TH-IR cell groups underwent some modifications during the period studied, its general pattern remained relatively stable after E12. This contrasted with the sequential appearance of the 5-HT-IR cell groups and their spatial transformations during this period. Using the rhombencephalic isthmus as a landmark, we found that conspicuous 5-HT-IR fibre bundles penetrated into the mesencephalon from E13 onwards, but that the 5-HT IR cell bodies were exclusively located caudal to the borderline between the mesencephalon and the rhombencephalon (the rhombencephalic isthmus). We therefore suggest the term "rostral rhombencephalic raphe nuclei" for the rostral 5-HT cell groups instead of "mesencephalic raphe nuclei," which is a misnomer. Close spatial association between TH and 5-HT-IR elements was observed mainly in the caudal rhombencephalon, where 5-HT-IR fibres coursed through an area containing numerous TH-IR cell bodies (the presumed anlagen of groups A1-2 and C1-3).

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The GLUT4 glucose transporter is primarily expressed in skeletal muscle, heart and adipose tissue, where its expression is postnatal, coincident with the acquisition of insulin-regulated glucose transport. In muscle, contraction also regulates GLUT4 activity in the postnatal animal. Here we demonstrate that GLUT4 is expressed in the developing mouse embryo with specific tissue and spatiotemporal patterns. From embryonic day 9 (E9; E1 = day of copulation plug) to postnatal day 70 (P70), mice were analyzed for GLUT4 mRNA and protein expression by in situ hybridization, immunohistochemistry and immunoblot. Specificity was confirmed with sense riboprobe hybridization and peptide competition, respectively. At E9, GLUT4 was detected in the cranial neural folds in the outer (mantle) layer of the neuroepithelium. At E10, expression was present throughout the developing heart and was prominent in the endocardial cushions through E12. At E10–12, GLUT4 was also prominent in craniofacial mesenchyme. GLUT4 expression in cartilage and bone was evident at E12 and was maintained throughout early postnatal life. GLUT4 was apparent throughout embryonic development in the ventricular epithelium, choroid plexus and in the developing cerebellum. At birth, cardiac expression was reduced and GLUT4 was most evident in cartilage, bone and specific brain regions. In the latter, GLUT4 expression was most evident in the cerebellum, specifically in the external granular layer through P7 and in the internal granular layer thereafter. Maximal GLUT4 protein levels in the cerebellum were measured between P14 and P21 and were reduced in the adult brain. These findings suggest that GLUT4-mediated glucose transport may play important roles during development of the brain and nonneuronal tissues in the mouse embryo.