Ultrastructure And Neurotransmitte Distribution Of Intestinal Nerve Of Remak In Chicken

Enteric nerve system(ENS), distributing in the intestine wall of mammals, is the third part of autonomic nerve system and independents from the central nervous system. The ENS of avian contains neurons that are intrinsic to the gastrointestinal tract, as well as axons of the intestinal nerve of Remak(INR). The sacral neural crest cells migrate and form INR. The INR is unique to birds, commencing at the jejunal-duodenal border, it traverses the mesentery and mesorectum running parallel to the gut until terminating at the cloaca. The INR innervates not only the intestine, but also the ovaries, the bursa of Fabricius, the didymus, the magnum and shell gland of the oviduct. In present study, electron microscopic studies, immunohistochemistry and in situ hybridization histochemistry was undertaken to determine the components and charaters of INR.ExperimentⅠUltrastructure of intestinal nerve of Remak in chicken In comparison with investigations in mammals there is very little information about the ultrastructures of avian autonomic nerves. Electron microscopy studies have been made on some autonomic nerve in the chicken and comparisons made with homologous structures in mammals. However, the INR in bird, that has no mammalian homologue. An electron microscopy study was undertaken to determine the ultrastructure of INR in chicken in present study. The ganglia of INR were composed of the cell bodies of neurons, satellite cells, myelinated and unmyelinated nerve cell processes, plasma cells and blood capillaries. The neuronal profiles had irregular shapes, each neuron cantained a single nucleus. The shape of the nuclei varied, and the nuclei were smoothsurfaced or the surfaces were furrowed to varying degrees. Parallet stacks of rough endoplasmic reticulum were often seen in the peripheral regions of the cell bodies. The satellite cells were smaller than neurons, and almost surrounded the cell bodies of neurons. There kinds of varicoses or terminals of processes were found in the ganglion of INR. The first kind varicoses or terminals contained numerous SSV and very few LGV, the second kind contained equal number of SSV and LGV, and the third kind contained many cytochondriomes and varying numbers synaptic vesicles. There were also two kinds of synapses, GrayⅠand GrayⅡ, been found in the ganglion of INR. The unmyelinated axons are obviously more than myelinated axons in the nerve trunk between the ganglia of INR. So, perhaps the ultrastructural characteristic of chicken INR correlated with the complicated functions of INR.ExperimentⅡDistribution of choline acetyltransferase and dopamine hydroxylase in intestinal nerve of Remak INR is unique to birds, and the characters are unknown till now. In present study, the morphology and distribution of ChAT and DβH neurons were examined by SABC immunohistochernistry reactions. The results showed that the percentages of ChAT and DβH neurons in the Remak nerve were 51.74±9.00% and 36.21±8.81% respectively. The morphologies of the positive neurons were both polygon with several protrusions and oval with one protrusion. These neurons located between bundles of nerve fibres. Several DβH neurons were observed in nerve trunk between ganglions. Some nerve fibres were ChAT or DβH positive. It is suggested that Remak nerve in the chicken is a mixed nerve which contains both parasympathetic postganglionic and sympathetic postganglionic neurons, and the proportion is about 52% and 36% respectively.ExperimentⅢPreparation of chicken vasoactive intestinal peptide RNA probe and its detection in intestinal nerve of Remak by in situ hybridization histochemistry To study the distribution of vasoactive intestinal peptide(VIP) mRNA in chicken INR, the sense and anti-sense digoxigenin(DIG) labeled RNA probe were prepared and utilized. The fragment of VIP gene was obtained by RT-PCR through total RNA of chicken brains. Amplified cDNA fragment was subcloned into pGM-T Easy vector, and the plasmid was transformed into E. coli DH5a and chosen by"white-blue plaque selection". The recombinant plasmid was identified by EcoRⅠrestriction enzyme digestion and sequencing, then VIP/pGM-T Easy vector was linearized with the restriction enzyme of NcoⅠand SalⅠrespectively. The sense and anti-sense DIG labeled RNA probes were producted by SP6 and T7 RNA polymerases respectively and transcription in vitro according to the protocol of"DIG RNA Labeling Kit(SP6/T7)". Certificated by dot blot hybridization, the sense and anti-sense RNA probes were prepared successfully. The distribution of VIP-mRNA in chicken INR was examined by in situ hybridization histochemistry(ISHH). Plenty of VIP mRNA positive neurons were found in the INR. Of these labeled neurons, 74.71±12.02% and 86.60±9.63% VIP mRNA positive neurons were distributed in juxta-jejunoileum and juxta-rectal portion of INR respectively. Some fibers of INR were weakly positive. In conclusion, the sense and anti-sense DIG labeled RNA probes for ISHH of VIP were prepared successfully in this experiment, which provided an approach to study further the location of VIP-mRNA in nerve tissue; the result of ISHH suggested the existence of VIP-mRNA in neurons of chicken INR.ExperimentⅣPreparation of chicken somatostatin precursor 1 RNA probe and its detection in intestinal nerve of Remak by in situ hybridization histochemistry In order to study the distributions of somatostatin precursor 1(PSS1) mRNA in chicken INR, the sense and anti-sense digoxigenin(DIG) labeled RNA probes were prepared. The distributions of PSS1 mRNA in the INR were examined by means of in situ hybridization histochemistry(ISHH). Plenty of PSS1 mRNA positive neurons were found in the INR. Of these labeled neurons, 86.98±7.93% and 86.07±6.11% PSS1 mRNA positive neurons were distributed in juxta-jejunoileum and juxta-rectal portion of INR respectively. Most of the fusiform or oval labeled neurons distributed in verge of the ganglia in small groups, some of the labeled neurons were also found in the nerve trunk between ganglia. In conclusion, PSS1 mRNA were transcribed in most of the INR neurons. We also discussed the possible innervation of these positive neurons to the intestinal and oviductal wall.ExperimentⅤPreparation of chicken preproenkephalin RNA probe and its detection in intestinal nerve of Remak by in situ hybridization histochemistry Enkephalin is a kind of very important peptide, but the distribution of enkephalin in INR is unclear. In order to study the distributions of preproenkephalin(PPE) mRNA in chicken INR, the sense and anti-sense digoxigenin(DIG) labeled RNA probes were prepared. The distribution of PPE mRNA in the INR was examined by means of in situ hybridization histochemistry(ISHH). Plenty of PPE mRNA positive neurons were found in the INR. Of these labeled neurons, 83.79±7.96% and 96.04±4.53% PPE mRNA positive neurons were distributed in juxta-jejunoileum and juxta-rectal portion of INR respectively. Most of the fusiform or oval labeled neurons distributed in verge of the ganglia in small groups, some of the labeled neurons were also found in the nerve trunk between ganglia. In conclusion, PPE mRNA were transcribed in most of the INR neurons. We also discussed the possible innervation of these positive neurons to the intestinal and oviductal wall. It is strongly supported that the VIP mRNA, the PSS1 mRNA and PPE mRNA are co-expressed in all the INR.