Immunohistological Detection Of Acetylcholine And Serotonin In The Different Development Phase Of Arenicola Brasiliensis (Polychaeta)

Acetylcholine (ACh) generally exists in most of the animals, regulating the function of animal's body nerve conduction, muscle movement, feelings and digestion. During the animal reproduction and development process, ACh also play an important role in regulation about the eduction of eggs and sperm, combination of egg binding, information transfer in intracellular and intercellular in the cleavage stage and subsequent growth process. 5-HT as a neurotransmitter and gastrointestinal hormones widely distributed in vertebrates and invertebrates body, play an important regulatory role in biological reproductive, perceptional and behavioral functions. Besides, 5-HT in earlier form of animal body development as a kind of growth signal, control the development of the gametophyte, process of early merogenesis and differentiation of cells. This paper adopts immunohistochemical S-ABC method (strept avidin-biotin complex), to locate the distribution of AChE, M1 mAChR, M3 mAChR,α1 nAChR, 5-HT and 5-HTR 1A in Arenicola brasiliensis embryos, larvae, the body wall and its appurtenances, digestive system and nervous system.The result of Arenicola brasiliensis embryos and larvae immunohistochemical positioning shows that AChE, M1 mAChR, M3 mAChR,α1 nAChR, 5-HT and 5-HTR 1A were positivity stained from unfertilized egg to 5-setiger larvae, but both the density and degree of distribution of different substances in the same growth phase, the same substance at different development stages were in different ways. Overall, the distribution of AChE and ACh receptors, 5-HT and 5-HTR 1A, AChE and 5-HT showed similar rules, and the distribution site of strong positive immune-reaction roughly the same. Positive immune-reaction in unfertilized more focused on cell membranes, the surrounding cytoplasm and perinuclear cytoplasm. Positive reaction of fertilized eggs more focused on the nuclear area, cell membranes and the surrounding cytoplasm. At the cleavage stage positive reaction mainly distributed in cell membranes and nuclear round about. The positive reaction in the ectoderm is stronger than the endoderm in the phase of gastrulae. At the Larva stage they were mainly detected in the head region, digestivetract, the areas where cilia grew, and setae etc. The widely distribution of positive immune-reaction in Arenicola brasiliensis embryos and larvae indicate that ACh, 5-HT may act together as important regulator during Arenicola brasiliensis development. The results of immunohistochemical S-ABC staining method showed that AChE, M1 mAChR, M3 mAChR,α1 nAChR, 5-HT and 5-HTR 1A were widely exist in the body of Arenicola brasiliensis, but different in rules. The body wall of Arenicola brasiliensis is composed of epithelium, circular muscle and longitudinal muscle. AChE, M1 mAChR, M3 mAChR,α1 nAChR, 5-HT and 5-HTR 1A were all showed positive reaction in the body wall, and positive material was centralized in the dissociative sides of the epithelium. But the reaction of M3 mAChR is weaker than the others. The appurtenances of the body wall consisted of gill, parapodia, chaetae and metanephridium. Gill is the respiratory organ of the Arenicola brasiliensis, the quantity of these neurotransmitters was high in the epithelium cells of the gill, and positive material was observed on the dissociative side of the gill. Parapodia and chaetae are the motor organs of the Arenicola brasiliensis. In the parapodia, both the epithelium cells and parapodia muscle were showed positive reaction, but the reaction ofα1 nAChR is weaker than the others. In the chaetae, these neurotransmitters were all showed strong positive or positive reaction. Metanephridium is the excretory organ of the Arenicola brasiliensis. The epithelium cells of metanephridium showed AChE, M1 mAChR, M3 mAChR,α1 nAChR, 5-HT and 5-HTR 1A strong positive or positive reaction, and positive material of these neurotransmitters were observed on the dissociative side of the epithelium.The digestive system of Arenicola brasiliensis was mainly composed of oesophagus, oesophageal pouch, stomach, intestine and rectum. The wall of the digestive tract consisted of three layers: tunica mucosa, tunica submucosa and adventitia from the inside to the outside. Immunohistochemical staining method showed that there were AChE, M1 mAChR, M3 mAChR,α1 nAChR, 5-HT and 5-HTR 1A immunoreactive positive cells and positive materials in the digestive tract, but the number and distribution of positive cells and positive materials were not the same. As a whole, strong positive cells were mostly observed in the tunica mucosa and adventitia, and positive material was observed on the dissociative side of tunica mucosa. Tunica submucosa showed weak positive reaction. The Nervous system of Arenicola brasiliensis consisted of suprapharygeal ganglion, circumpharygeal connective, subpharygeal ganglion and ventral nerve cord. The cerebral ganglion was divided into two parts as cerebral cortex and medullary layer. The AChE, M1 mAChR, M3 mAChR,α1 nAChR, 5-HT and 5-HTR 1A immunoreactive positive cells and nerve fibers were present in the cerebral cortex and medullary layer of cerebral ganglion, while the strong positive cells mainly distributed in cerebral cortex, and the nerve fibers showed positive or weak positive reaction. At the same time, the distribution of M3 mAChR andα1 nAChR in the subpharygeal ganglion was stronger than the oter areas. It could be presumed that neurotransmitters might regulate several physiological functions, such as digestion, motion, and so on. The different density of neurotransmitters in different parts of Arenicola brasiliensis might correlate to their various functions.