Mary Beth Wilkie

Cytokine regulation of embryonic rat dopamine and serotonin neuronal survival in vitro

Interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), and tumor necrosis factor‐α (TNF‐α) 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‐1β, IL‐6, and TNF‐α 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‐1β and TNF‐α 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‐α. 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‐1β, IL‐6, and TNF‐α 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 of 5-HT2A, 5-HT2B and 5-HT2C receptors in the mouse embryo

Expression patterns of 5‐HT2A, 5‐HT2B and 5‐HT2C receptors during mouse embryogenesis were investigated using highly specific monoclonal antibodies. Differential and overlapping spatio‐temporal patterns of 5‐HT2A, 5‐HT2B and 5‐HT2C 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‐HT2 receptor antagonists for 2 days in whole embryo culture. The most potent was the pan 5‐HT2 receptor antagonist ritanserin, which has high affinity for the 5‐HT2B receptor. Ritanserin caused 100% malformed embryos at a dose of 1 μM. The 5‐HT2A/2C receptor antagonist mianserin also caused a significant number of malformed embryos, but only when used at a 10 fold higher dose (10 μM). Ketanserin, which primarily targets 5‐HT2A 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‐HT2 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.

Brain growth retardation due to the expression of human insulin like growth factor binding protein-1 in transgenic mice: an in vivo model for the analysis of igf function in the brain

Three lines of transgenic (Tg) mice carrying a fusion gene linking the mouse metallothionein-I promoter to a cDNA encoding human insulin-like growth factor binding protein-1 (hIGFBP-1) were found to express the transgene in brain. As judged by comparing Tg brain weights to those of non-transgenic littermates, adult hemizygotic Tg mice of each line exhibited brain growth retardation (16.2%, 14.4% and 8.1% reductions in weight, respectively in each line). In two lines, total brain DNA and protein content were decreased. Further analysis indicated that the brain growth retardation was manifested in the second week of postnatal life. Given that the insulin-like growth factors (IGFs) stimulate cell proliferation and/or survival in neural cultures and that hIGFBP-1, when present in a molar excess, inhibits IGF interactions with their cell surface receptors, the brain growth retardation in hIGFBP-1 Tg mice likely results from hIGFBP-1 inhibition of IGF-stimulated growth-promoting actions. These hIGFBP-1 Tg mice should prove useful in defining IGF actions during postnatal brain maturation.

Combined serotonin immunocytochemistry and 3H-thymidine autoradiography: in vivo and in vitro methods.

Principle of Technique This method was devised to facilitate studies of developing neurons of known transmitter content, as defined by immunocytochemistry, in terms of their state of differentiation at the time of transmitter synthesis (proliferating or nonproliferating, Figure 2) as well as their relationship to other less differentiated precursor cells in their vicinity (Figures 3, 4). We have specifically applied this methodology to studies of the developing 5-hydroxytryptamine (5-HT) system in the embnyonic and postnatal rat brain as well as in dissociated cell cultures of 5-HT neurons (5-8,14), in an attempt to provide answers to questions raised in our previous studies with regard to pnetransmission functions for neurotnansmitters in development (2-4).

Prenatal Expression of the GLUT4 Glucose Transporter in the Mouse

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.