Glial cell line-derived neurotrophic factor is a key neurotrophin in the postnatal enteric nervous system

Abstract

The enteric nervous system (ENS) continues its structural and functional growth after birth, with formation of ganglia and the innervation of growing smooth muscle. However, little is known about factors in the postnatal intestine that influence these processes. We examined the presence and potential role of glial cell line-derived nerve growth factor (GDNF) in the rat postnatal ENS using neonatal tissue, primary co-cultures of the myenteric plexus, smooth muscle, and glial cells as well as cell lines of smooth muscle or glial cells. Western blot analysis showed that GDNF and its co-receptors rearranged during transfection (RET) and GDNF family receptor alpha-1 were expressed in the muscle layer of the neonatal and adult rat intestine. Immunohistochemistry localized the receptors for GDNF to myenteric neurons, while GDNF was localized to smooth muscle cells. In a co-culture model, GDNF but not nerve growth factor, brain derived neurotrophic factor or neurotrophin-3 significantly increased neuronal survival and more than doubled the numbers of neurites in vitro. RT-PCR, qPCR, Western blotting, ELISA, and immunocytochemistry as well as bioassays of neuronal survival and of RET phosphorylation all identified intestinal smooth muscle as the source of GDNF in vitro. GDNF also induced morphological changes in the structure and organization of neurons and axons, causing marked aggregation of neuronal cell bodies and collinear development of axons. As well, GDNF (50-150 ng mL(-1)) significantly increased [(3)H]-choline uptake and stimulated [(3)H]-acetylcholine release. We conclude that GDNF derived from intestinal smooth muscle cells is a key factor influencing the structural and functional development of postnatal myenteric neurons.