3.03 TNF-related protein LIGHT reduces BDNF-dependant neuronal growth in sensory neurons Nuria Gavalda, Alun M Davies Cardiff School <strong>of</strong> Biosciences,, Biomedical Sciences Building 3,, Museum Avenue,, Cardiff CF10 3US,, Wales, UK LIGHT is a protein belonging to the TNF superfamily that was originally identified as a weak inducer <strong>of</strong> apoptosis. LIGHT has been shown to be involved in the costimulation and homeostasis <strong>of</strong> T-cells, however, no data exist that describe a function for this ligand during the development <strong>of</strong> the nervous system. Our present work reveals a novel role for LIGHT in the regulation <strong>of</strong> neurite growth during the development <strong>of</strong> mouse sensory neurons. LIGHT is capable <strong>of</strong> binding to both the lymphotoxin-beta (LTβR) and HVEM receptors, both <strong>of</strong> which were expressed by nodose neurons. Nodose neurons that were transfected with a plasmid encoding LIGHT had significantly reduced neurite growth, during a specific period <strong>of</strong> embryonic through to postnatal development. The activation <strong>of</strong> several signalling pathways have been shown to mediate the effects <strong>of</strong> LIGHT in T-cells. These include NF-κB, JNK and ERK/MAPK. In our cellular model, only ERK/MAPK is activated by LIGHT, but not NF-κB nor JNK. Specifically blocking the LIGHT induced ERK activation with the selective inhibitors, U0126 and PD98059, inhibits the ability <strong>of</strong> LIGHT to affect neurite growth in nodose neurons. In contast to this, inhibiting the NF-κB pathway using specific NF-κB mutant proteins or the JNK pathway, using specific JNK inhibitor, had no effect on the ability <strong>of</strong> LIGHT to affect neurite growth in nodose neurons. All together, these results show a novel role <strong>of</strong> LIGHT in sensory neuron development. 3.04 Competitive, intrinsic and stochastic determinants <strong>of</strong> neuromuscular synapse elimination in transgenic YFP expressing mice Teriakidis A, Jenkins N, Willshaw D J, Ribchester R R Centres for <strong>Neuroscience</strong> Research and Neuroinformatics DTC, University <strong>of</strong> Edinburgh Competition at motor endplates strongly influences elimination <strong>of</strong> polyneuronal innervation in neonatal muscles but it is still not known whether intrinsic properties <strong>of</strong> motor neurones also determine ultimate motor unit size. We are using transgenic mice expressing fluorescent protein in motor neurones to measure and model the changes in motor unit size as neuromuscular synapses form or become eliminated. First we designed experiments to test whether motor unit size in adult lumbrical muscles becomes reduced even when all other competing motor units are removed, by partial denervation at birth (neonates anaesthetised by chilling). We found that motor unit size was not reduced when these mice reached adulthood, suggesting that competition is sufficient to account for synapse elimination during development. Next we examined whether the initial motor innervation pattern is random. Analysis <strong>of</strong> YFP-expressing motor units in lumbrical muscles <strong>of</strong> thy1.2-YFPH mice indicated that all connections made by a motor neurone are restricted to a single lumbrical muscle with no divergence between muscles. Stochastic modeling suggested that a key indicator <strong>of</strong> randomness is initial converge <strong>of</strong> branches <strong>of</strong> the same axon on single motor endplates. To test this we examined adult muscles in the earliest stages <strong>of</strong> reinnervation (12-14 days) after nerve crush under halothane/N20 anaesthesia. We found several compelling instances <strong>of</strong> within-unit convergence on motor endplates. Thus, stochastic properties play an important role in establishing the divergent and convergent innervation pattern within muscles but competitive interactions at polyinnervated junctions are more important than intrinsic properties <strong>of</strong> neurones during synapse elimination. 3.05 Investigation into the differentiation <strong>of</strong> motor neurons derived from human pluripotent stem cells and assessment <strong>of</strong> their function in vitro Pan C, Przyborski S School <strong>of</strong> Biological and Biomedical Science, Durham University, South Road, Durham DH1 3LE It is understood that retinoic acid (RA), sonic hedgehog (Shh) and bone morphogenic proteins (BMPs) play an important role in cell fate determination and the specification <strong>of</strong> inter-neurons and motor neurons along the dorsal-ventral axis in the neural tube. In this study, we propose to evaluate the function <strong>of</strong> these signalling molecules to instruct the differentiation <strong>of</strong> human pluripotent stem cells. TERA2.cl.SP12 embryonal carcinoma (EC) cells are a robust caricature <strong>of</strong> human embryogenesis and an accepted model <strong>of</strong> neural differentiation. Gene and protein expression analyses indicate that human EC cells respond to RA, BMPs and Shh in a conserved manner and regulate neural transcription factors and structural proteins in a predicted way as cells commit toward the motor neuron phenotype. To assess the function <strong>of</strong> these differentiated neurons, we tested their ability to innervate skeletal muscle myotubes and induce contraction. We showed that muscle contraction could be manipulated pharmacologically: curare and atropine blocked myotube contraction, whereas acetylcholine and carbachol increased the number <strong>of</strong> contractile events. In other experiments, we have also shown that cells exposed to RA and Shh in conjunction with other growth factors over different time periods, preferentially form oligodendrocytes and/or interneurons. These results indicate it is feasible to control and direct the differentiation <strong>of</strong> human stem cells and produce specific neuron subtypes in vitro as a model to investigate the molecular mechanisms and signalling pathways that control these processes in man. 3.06 Clonally derived adult stem cell-derived soluble factors and their instructive effects on neural stem and progenitor cells Emmerson R, Cr<strong>of</strong>t A, Przyborski S A School <strong>of</strong> Biological and Biomedical Sciences, University <strong>of</strong> Durham, South Road, Durham, DH1 3LE; ReInnervate Ltd, Durham, DH1 3HP Transplantation <strong>of</strong> both mesenchymal stem cells (MSCs) and dermal cells has been shown to result in functional improvement in animal models <strong>of</strong> neurological disease, however, the mechanism by which this occurs remains unclear. We have shown that MSCs produce factors that instruct neural stem cells (NSCs) to adopt neuronal and glial lineages, and this is dependent on the developmental status <strong>of</strong> the MSC. MSCs induced to form cellular aggregates, express neural antigens such as nestin and GFAP, and in co-culture studies instruct NSCs to adopt a predominantly neuronal fate. We have clonally derived MSC cell lines that express neural markers such as Tuj-1 and GFAP without induction <strong>of</strong> cell aggregates. Therefore clonal MSCs expressing neural antigens could have a similar instructive effect on NSCs as their parent MSC population. Induction <strong>of</strong> neural antigen expression such as nestin and Tuj-1 has also been demonstrated in dermal populations <strong>of</strong> stem cells. The dermal papilla (DP) region is thought to be a niche <strong>of</strong> dermal cells able to express neural proteins. Clonal cell lines derived from the DP and the dermal sheath (DS) form aggregates that are similar to both those derived from dermal stem cells and induced MSCs. We are investigating whether dermal aggregates could have an instructive effect on NSCs that is similar to MSCs. The study <strong>of</strong> clonally derived hair follicle and MSC populations will enable a better understanding <strong>of</strong> the effects <strong>of</strong> soluble factors produced by adult stem cell populations on neurogenesis. Page 2/101 - 10/05/2013 - 11:11:03
3.07 Congenital hypothyroidism alters formalin-induced pain response in neonate rats Behzadi G, Mohamad R <strong>Neuroscience</strong> Research Center and Physiology Department, Fac. Med., Shaheed Beheshti Med Sci Univ. POBox 19835-181 It is known that postnatal maturation <strong>of</strong> the CNS is critically dependent on the thyroid hormone levels. The present study attempted to investigate the development <strong>of</strong> nociceptive response following induction <strong>of</strong> the formalin pain in congenitally hypothyroid pups during the first three postnatal weeks. Time pregnant rats received PTU from 16th day <strong>of</strong> gestation until 23rd postnatal day. Control and <strong>of</strong>fspring pups were received different formalin solution in right hind paw at 7, 15, and 23 days <strong>of</strong> age. Significant reduction in weight gain was evident in PTU-treated <strong>of</strong>fspring from postnatal days 15 up to 23 (P
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