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Ca2+ influx through ionotropic glutamate receptors regulates a variety of developmental processes including neurite outgrowth and naturally occurring cell death. In the CNS, NMDA receptors were originally thought to be the sole source of Ca2+ influx through glutamate receptors; however, AMPA receptors also allow a significant influx of Ca2+ ions. The Ca2+ permeability of AMPA receptors is regulated by the insertion of one or more edited GluR2 subunits into the receptors. Although Ca2+-permeable AMPA receptors are a familiar feature in developing neurons, the developmental function of these receptors during the formation of the nervous system has yet to be established. This study was designed to investigate the expression and functional role of Ca2+-permeable AMPA receptors in developing chicken spinal motoneurons. Our results demonstrate that chicken lumbar motoneurons express functional AMPA receptors as early as embryonic day (E) 5. Electrophysiological recordings of kainate-evoked currents indicate a significant reduction in the Ca2+ permeability of AMPA receptors between E6 and E11. During this developmental period, the Ca2+ permeability of AMPA receptors decreases three-fold. Reduction in the Ca2+ permeability of AMPA receptors is accompanied by increased expression of GluR2 mRNA in the spinal motoneuron pool. Changes in GluR2 mRNA expression occur in parallel to changes in GluR2 protein expression in the chicken ventral spinal cord. Changes in the Ca2+-permeability of AMPA receptors are not mediated by age-dependent changes in the editing pattern of GluR2 subunits. At early stages of development, functional AMPA receptors were composed of a combination of GluR3 and GluR4 subunits. mRNA analysis indicates that GluR4 is the most abundant subunit in the chicken ventral spinal cord between E6 and E11. Immunohistochemistry analysis of spinal cord sections also demonstrated that both GluR3 and GluR4 proteins are expressed at E6 and E11. Expression of Ca2+-permeable AMPA receptors regulates the maturation of dendritic outgrowth in developing spinal motoneurons. Measurements of dendritic length and branching pattern demonstrate significant changes in the dendritic morphology of spinal motoneurons between E6 and E11. Blockade of AMPA receptor activation with CNQX between E5 and E8 causes a significant increase in dendritic outgrowth in lumbar motoneurons, when compared with vehicle-treated embryos. Treatment of chicken embryos with CNQX between E8 and E11, when AMPA receptors become Ca2+-impermeable, has no affect on dendritic morphology. However, blockade of NMDA receptor activation with MK-801 causes a significant reduction in dendritic outgrowth of lumbar motoneurons by E11. These findings indicate that AMPA receptor activation between E5 and E8 limits dendritic outgrowth in developing motoneurons, whereas NMDA receptor activation is involved in dendritic remodeling after the establishment of synaptic contacts with sensory afferents.

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No description available.

No description available.

The glial cell-line derived neurotrophic factor (GDNF) and neurturin (NTN) belong to a structurally related family of neurotrophic factors. NTN exerts its effect through a multi-component receptor system consisting of the GDNF family receptor alpha 2 (GFRα2), proto-oncogene RET and/or NCAM. GFRα2 is spliced into at least three isoforms, GFRα2a, GFRα2b and GFRα2c. The present study investigated the expression and functional differences of GFRα2 isoforms. These receptor isoforms are differentially expressed in specific human brain regions. Using Neuro2A model, GDNF and NTN promote neurite outgrowth via GFRα2a and GFRα2c, but not GFRα2b. These GFRα2 isoforms regulate different early response genes when stimulated with GDNF and NTN. Interestingly, using co-expression models, GFRα2b inhibits ligand induced neurites outgrowth of GFRα2a and GFRα2c, and also the related receptor, GFRα1a. More intriguingly, ligands activated GFRα2b was also able to attenuate neurite extension induced by an unrelated stimulation using retinoic acid. MAPK activation induced by GDNF was not attenuated by GFRα2b in a co-expression model, while the early response genes expression profile (up-regulation of FosB) was similar to that induced by GFRα2b alone. This study suggest that GFRα2b is not merely a dominant negative isoform, but signals through a yet to be determined mechanism to antagonize and inhibit neuritogenesis. Together, these data suggest a new paradigm for the regulation of growth factor signaling and neurite outgrowth via an inhibitory splice variant of the receptor. / Singapore-MIT Alliance (SMA)

The present study was undertaken to evaluate the effects of ultra low concentrations (10-9 or 10-14 M) of morphine on neurite elongation in cultured neurons dissociated from rat spinal cords and cerebral cortex. In fetal serum (FS) or fetal serum-free supplemented with cAMP media, the length of longest neurite was significantly increased by 10-9 or 10-14 M morphine. For example, 10-14 M morphine increased neurite length by 24±0.5% and 27±0.3% in spinal cord neurons, and 18±0.2% and 17±0.6% in cortical neurons. Morphine (10-6 M) had no significant effect on neurite length of spinal and cortical neurons. The relative frequency distribution of neurite length revealed 61±2.7% of spinal neurons and 48±2.6% of cortical neurons are responsive to ultra low concentrations of morphine. In the responsive populations, morphine (10 -14 M) enhanced the neurite outgrowth in spinal neurons by 58±0.9% and 48±1.2% and in cortical neurons by 31±0.6% and 28±0.9% in FS and cAMP-supplemented media, respectively. Pretreatment with naloxone did not prevent the morphine effect. The result shows that morphine at ultra low concentrations enhances neurite outgrowth of spinal and cortical neurons via a naloxone-independent mechanism.

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No description available.

Several uterine-derived paracrine factors have been implicated as critical regulators of conceptus development in cattle, but it remains unclear how these factors work together to establish and maintain pregnancies. The primary objectives of this work were to establish if cooperative interactions between epidermal growth factor (EGF), fibroblast growth factor-2 (FGF2) and insulin-like growth factor-1 (IGF1) promote bovine trophoblast cell proliferation, and to decipher the intracellular signaling mechanisms employed by these growth factors to regulate cell proliferation. Pilot studies established effective concentrations for each growth factor on a bovine trophoblast cell line (CT1). The first set of studies examined how each factor worked individually or in conjunction with each other to impact CT1 proliferation. Mitotic index (percentage of EdU-positive nuclei after a 45 min challenge) was increased (P<0.05) by supplementation with 10 ng/ml EGF, 10 ng/ml FGF2, or 50 ng/ml IGF1 when compared with non-treated controls. In addition, a greater increase (P<0.05) was detected when all three factors were supplemented together. A follow-up study determined that supplementation of any two growth factors could not replicate the cooperative effect noted when all three factors were provided. A second set of studies was undertaken to examine how mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/AKT (PI3K/AKT) signaling systems mediate the independent and cooperative effects of these paracrine factors. Both EGF and IGF1 transiently activated mitogen-activated protein kinase3/1 (MAPK3/1) in CT1 cells as determined by Western Blot analysis. By contrast, FGF2 did not affect MAPK3/1 phosphorylation status, but increased AKT phosphorylation status. Neither EGF nor IGF1 impacted AKT activity. Supplementation with a pharmacological inhibitor of MAPK3/1 (PD98059) prevented EGF-, IGF1-, and FGF2-dependent increases in CT1 cell proliferation. This inhibitor also completely abolished the increases in cell proliferation observed when all three factors were supplemented together. Supplementation with a pharmacological inhibitor of AKT (Wortmannin) reduced FGF2-stimulated CT1 proliferation, but did not impact EGF- and IGF1 effects. The AKT inhibitor partially attenuated the cooperative effects of all three factors on CT1 cell proliferation. A final study examined how the combination of EGF, FGF2, and IGF1 affect bovine embryo development. In vitro produced bovine blastocysts were cultured either with the combination of growth factors or vehicle only from day 8 to day 12 post-in vitro fertilization (IVF). The combination of EGF, FGF2, and IGF1 increased (P<0.05) the percentage of hatched blastocysts and outgrowth formation versus controls. Increased (P<0.05) diameters were detected in blastocysts treated with the combination of three growth factors on day 12 post-IVF when compared to controls. Treatment with the combination of EGF, FGF2, and also IGF1 increased (P<0.05) the change of diameter from day 8 to 12 post-IVF. In summary, these observations provide evidence that cooperative interactions of uterine-derived factors promote trophoblast proliferation and conceptus development in ways that may promote the establishment and maintenance of pregnancy in cattle. The mechanisms utilized for these activities remain unresolved, but MAPK3/1 and PI3K/AKT signaling systems appear to play integral roles in some of these processes. / Master of Science