GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR MODULATES STRUCTURE AND FUNCTION OF POSTNATAL MYENTERIC NEURONS
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The enteric nervous system (ENS) continues its development after birth, with formation of ganglia and functional synapses; plasticity is also demonstrated in significant axon growth that occurs after experimental colitis in the adult colon. However, little is known about factors in the postnatal intestine that influence and regulate these processes. Therefore we tested the effects of known neurotrophins, NGF, NT-3, BDNF and GDNF on neonatal rat myenteric neurons. Cocultures were developed by isolating the myenteric plexus and surrounding muscular wall from neonatal rats, and effects of exogenous treatment of neurotrophins were analyzed using immunocytochemistry and image analysis. Western blotting and immunocytochemistry were performed to detect implicated neurotrophins and their receptors in the postnatal intestine. Functional aspects of effects of implicated neurotrophins were assessed by [3H]choline uptake and acetylcholine release in myenteric neurons. Last, TNBS-colitis was induced in adult rats to determine changes in GDNF secretion during the course of the disease. Application of 100ng/mL GDNF to a neonatal intestinal coculture containing neurons, glia and smooth muscle cells produced a 91.5% (p≤0.05) increase in axons. GDNF induced morphological changes in the structure and organization of neurons and axons; the incidence of neurons present in ganglia increased by 11.2% (p≤0.05), with a 32.9% (p≤0.05) increase in aggregated axons. Western blotting and immunocytochemistry confirmed intestinal smooth muscle as the major source of GDNF and demonstrated the presence of the GDNF receptor complex, GFRα1 and RET in the myenteric plexus. Choline uptake significantly increased at 50, 100 and 150ng/ml doses of GDNF, whereas stimulated ACh release increased only at 100 and 150ng/ml doses. In TNBS-colitis, a decrease in 35kD GDNF at days 1 and 6 post-induction of inflammation was observed, with a concomitant increase in 15kD GDNF. We conclude that GDNF, produced by intestinal smooth muscle, is a key factor influencing development of the postnatal myenteric neuron and may play a role in ENS-restructuring post-inflammation.