Developmental regional distribution and neurochemical coding of enteric neurons in the zebrafish mutant lessen compared to the wildtype zebrafish

Abstracts / Autonomic Neuroscience: Basic and Clinical 163 (2011) 1–133 muscle recording. Histological analysis revealed changes in the mucosa, muscle, and neurons. The mucosal surface was sloughed off 3 hours following I/R but the epithelium appeared normal by 1 day in both nNOS KO and WT mice. More severe degenerative changes were observed in both the longitudinal and circular muscle layers of nNOS KO mice compared to WT mice (n = 3). Neutrophils were not present in the muscle layers and were infrequent in the mucosa of nNOS WT mice. However, neutrophil numbers increased in the longitudinal and circular muscle layers of nNOS KO animals (n = 3). Data to date indicate a reduction in the spontaneous muscle activity and amplitude of muscle contraction in response to electrical field stimulation (2, 5, and 20 Hz/60 V/100 pulses/0.5 ms), 3 and 24 hours following I/R. This work indicates that I/R causes severe longitudinal and circular muscle damage that may contribute to the dysmotility observed after intestinal I/R, and a possible protective role of NO that is reduced after nNOS knock-out. Keywords: ischemia, nitric oxide, intestine, nNOS, motility Financial support: National Health and Medical Research Council of Australia. doi:10.1016/j.autneu.2011.05.062 P.048 Developmental regional distribution and neurochemical coding of enteric neurons in the zebrafish mutant lessen compared to the wildtype zebrafish L. Uyttebroek (Laboratory of Human Anatomy and Embryology University of Antwerp), I. Shepherd (Department of Biology - Emory University, Atlanta), G. Hubens (Laboratory of Human Anatomy and Embryology - University of Antwerp), J.P. Timmermans (Laboratory of Histology & Cell Biology - University of Antwerp), L. Van Nassauw (Laboratory of Human Anatomy and Embryology - University of Antwerp) Hirschsprung's disease (HD) is a congenital disorder of the enteric nervous system characterized by aganglionosis along a variable portion of the colon. A recently developed mutant zebrafish, lessen, expressing some HD characteristics, is suggested to be an experimental model to unravel the underlying developmental mechanisms for HD. This study aims to compare the neurochemical coding of enteric neurons of the wildtype zebrafish with the mutant lessen to further validate this mutant as a suitable model for HD. Multiple immunofluorescence staining was used to detect previously characterized neurochemical markers at 3, 4 and 5 days post-fertilization (dpf) in the proximal (PI), mid (MI) and distal intestine (DI) of both wild type and mutant animals. In mutants, the number of enteric neurons was significantly reduced in DI (even absent at 3dpf) and MI, but less in PI at each embryonic stage. The proportion of nitrergic neurons was significantly reduced in all regions at 3 dpf, but nearly unaffected in PI and MI at 4 and 5dpf. Serotonin, calretinin and calbindin showed a delayed expression and a decrease in both number and proportion at all points of time and in each intestinal region. This study shows abnormalities in the number and the relative frequency of enteric neurons expressing various neurochemical markers at each embryonic stage. These results are similar as data obtained in the intestine proximal to the aganglionic segment and the aganglionic segment of the ls mutant mice, an experimental HD model, indicating that the lessen mutant is a suitable model to study HD. Keywords: enteric neurons, neurochemical coding, zebrafish, lessen mutant, Hirschsprung's disease 59 doi:10.1016/j.autneu.2011.05.063 P.049 Assessment quercetin supplementation in diabetic Wistar rats: Analysis of vasoactive intestinal polypeptide (VIP) immunoreactive myenteric neurons from jejunum J.V.C.P. Martins (Universidade Estadual de Maringá - Departamento de Ciências Morfológicas (DCM)), S.R.G. Souza (Universidade Estadual de Maringá - Departamento de Ciências Morfológicas), I. Zignani (Universidade Estadual de Maringá - Ciencias Morfológicas), C.G.S. Melo (Faculdade de Odontologia de Bauru, Universidade de São Paulo Anatomia), M.H. Miranda Neto, J.N. Zanoni (Universidade Estadual de Maringá - Ciencias Morfológicas) Aims: This study focus on investigate the harmful effects and the protective effects of the supplementation with quercetin on the neurons that express the vasoactive intestinal polypeptide (VIP) of the myenteric plexus of the jejunum of diabetic rats. Methods: Male Wistar rats (Rattus norvegicus) with 90 old-days were divided into four groups: normoglycemic (N); normoglycemic supplemented with quercetin (Q); diabetic (D); and diabetic supplemented with quercetin (DQ). The quercetin (200 mg/kg) was given orally for 16 wk in water (prepared daily). At 210-days-old animals were killed and their jejunum was collected and submitted to immunohistochemistry to VIP. Morphmetric analysis was performed in 2400 VIP-ergic varicosities for each group. Results: The supplementation with quercetin promoted a reduction (19,02%) in the varicosities area in the group Q when compared with the group N (p < 0,05). In group D, the mean area of the varicosities increased 24 % relative to group N (p < 0.05). The supplementation in group DQ recovered area of the varicosities to same values to those observed in group N (p > 0.05). Conclusion: This fact demonstrated that the quercetin prevented an increased of the varicosities in diabetic animals supplemented with quercetin, confirming a possible protective effect of this flavonoid, which can be explained by its anti-oxidant action on these myenteric neurons. Keywords: Diabetes mellitus, enteric neurons, jejunum, quercetin, VIP Financial support: Fundação Araucária. doi:10.1016/j.autneu.2011.05.064 P.050 Ileum of diabetic rats (Rattus norvegicus): Effect of supplementation with 2% L-glutamine on glial cells and myenteric neurons J.N. Zanoni, E.A. Tronchini, C.M. Tashima, E.P.B. Alves, R.V.F. Pereira (Universidade Estadual de Maringá - Departamento de Ciências Morfológicas) The effects of 2% L-glutamine supplementation on peripheral diabetic neuropathy and enteric glia in the ileum were investigated. Male Wistar rats were divided into the following groups: normoglycemics (N), normoglycemics supplemented with L-glutamine (NG), diabetics (D), and diabetics supplemented with L-glutamine (DG). For experimental supplementation of rats in groups NG and DG, Lglutamine was incorporated into the standard chow at a concentra-