Background and Significance Neuropeptide Y (NPY) is a peptide neurotransmitter shown to be a neuroproliferative factor in postnatal olfactory neurons. Alterations in NPY have been noted in diseases affecting the enteric nervous system such as diabetes. We investigated the role of NPY in enteric neuronal proliferation and suppression of apoptosis in a primary enteric neuronal culture as well as in an NPY knockout mouse model (NPY KO).
Methods Using a primary culture system of enteric neurons obtained by immunoselection from E14 rat embryonic intestines, we examined the effect of NPY on enteric neuronal proliferation (Brdu) and apoptosis (Hoechst and cleaved caspase 3 staining). NPY mediated phosphorylation of Akt was assessed by Western blot analysis. For the in vivo studies segments of proximal colon from WT and NPY KO mice were fixed and stained for peripherin (total number of myenteric neurons), NADPH diaphorase (nNOS-containing inhibitory neurons), and acetylcholine esterase (excitatory neurons). Electric field stimulation (EFS) of proximal colon strips was used to evaluate the inhibitory neuronal mediated relaxation of the colon.
Results Exposure of NPY (1 mM, 24 h) significantly increased proliferation of enteric neurons (% Brdu-positive neurons: vehicle: 37.6 6 4.3; NPY: 60 6 2, n = 4, p < .006). NPY also significantly reduced apoptosis in enteric neurons compared to controls assessed by the Hoechst (p < .002) and cleaved caspase 3 (p < .0001). The PI-3-K/Akt pathway has been demonstrated to be important in enteric neuronal survival. Enteric neurons cultured in the presence of NPY showed a significantly increased phosphorylation of Akt compared to vehicle and this was lost when treated with PI-3-kinase inhibitor LY294002 (p < .05 vs NPY, n = 3). We next examined the effects of NPY on enteric neurons in vivo. In NPY KO mice, there was a significant decrease in the number of inhibitory neurons and large fibers in the colon compared to WT mice (neurons: 0.01; large fibers: p = .0009, n = 6). No change was noted in the number of acetylcholine esterase staining excitatory neurons or large fibers (p = .3760; n = 6). EFS-evoked relaxation of the proximal colon was significantly impaired in NPY KO mice compared to WT mice (% relaxation at 48 V: WT: 40.94 6 4.6, NPY KO: 15.27 6 1.3, p = .002, n = 4).
Conclusions NPY promotes enteric neuronal survival and proliferation. Loss of NPY in the NPY KO mouse model is associated with a decrease in inhibitory neurons demonstrated by neuronal staining and by EFS recording from muscle strips. NPY can be a potential target for modulation of enteric neuronal survival.
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