Xeniou, Ourania; Romero, I.A. and Saffrey, M.J.
Role of neurotrophic factors (NTF) and extracellular matrix components (ECM) on VIP expression in the rat enteric nervous system (ENS).
In: Physiological Society Meeting, King's College London, December 2004, London, UK.
We studied changes in vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (nNOS) expression by myenteric neurons during early postnatal development in parallel with maturation of the environment of myenteric neurons, specifically the expression of ECM components and NTF. Although these molecules are known to promote neuronal differentiation [Shetty & Turner, 1998: Sieber-Blum et al. 1981], very little is known about their role in the early postnatal ENS. Sprague-Dawley rats were killed by cervical dislocation. The ileal muscularis externa, including myenteric ganglia, was processed for semi-quantitative RT-PCR at postnatal days P1, P7 and P21 (internal standards: βIII tubulin and PGP9.5). VIP (Fig. 1), nNOS and NT-3 mRNA levels showed a consistent increase between P1 and P21. Although no changes in GDNF mRNA levels were observed, there was an increase in levels of mRNA of its co-receptor, GFRα-1, indicating that the responsiveness of myenteric neurons to GDNF might be enhanced at older ages. Immunofluorescent labelling of ileal cross sections revealed changes in the levels of laminin (peak at P7) and fibronectin (peak at P21). Next, we tested the effect of ECM and NTF on myenteric neurons in cultures of dissociated myenteric ganglia (P7/8) [Schafer et al. 1997]. Changes in cell numbers per unit area of coverslips were measured after double labelling for βIII tubulin and VIP. GDNF, but not NT-3 or BDNF (all at 1 ng/ml), enhanced survival of the overall neuronal population (35-67% increase, n=3 experiments, P<0.02, paired t-test) and the percentage of VIP positive neurons (39-59%, n=3, P<0.02). Elevation of VIP and nNOS mRNA levels after treatment with GDNF was confirmed by RT-PCR. The effect of GDNF on neuronal survival was maintained in the presence of laminin (26-58% increase, n=3, P<0.05) and fibronectin (34-55%, n=3, P<0.012), although neither factor alone influenced neuronal survival. Fibronectin (10-18% increase, n=3, P=0.10), but not laminin (19-81% increase, n=3, P<0.04), reduced the effect of GDNF on the proportion of VIP positive neurons. Our study suggests that GDNF regulates VIP and nNOS expression by myenteric neurons. Fibronectin was found to influence the effect of GDNF on VIP phenotype. Together with the observed changes in the environment of myenteric neurons in the early postnatal gut, our results indicate that both NTF and ECM components are likely to contribute to postnatal development of the ENS.
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