The Differentiation Of Aromatic Amino Acid Hydroxylase And Monoamine Neurotransmitters In The Central Nervous System Of Nereis (Neanthes Japonica)

In this study,using the different polyclonal antibody by the Streptavidin Peroxidase(SP)immunohistochemical method and Colophony–Paraffin(CP)embedded section technique,combined with the rapid amplification of cDNA ends(RACE)technique,we described the expression and distribution of aromatic amino acid hydroxylases(AAAHs)including tryptophan hydroxylase(TPH),phenylalanine hydroxylase(PAH),tyrosine hydroxylase(TH),and dopamine-beta-hydroxylase(D?H),phenylethanolamine-N-methyltransferase(PNMT)in the central nervous system of Nereis Neanthes japonica(Polychaeta,Annelida),which was the typical model animal of low invertebrate Annelida.The relationship between AAAHs and monoamine neurotransmitters,and the gene evolutionary node of the AAAHs were analyzed.The important results and conclusions as follows:1.The results displayed strong 5-HT-like immunoreactivity(5-HT-IR),but weak or no TPH-like immunoreactivity(TPH-IR)with an extensive and consistent distribution pattern,by immunohistochemical staining of paraffin sections in the brain and ventral nerve cord of Neanthes japonica by using sheep anti-TPH polyclonal antibody and rabbit anti-5-HT antiserum.The neuron type,distribution,and projection of TPH-IR neurons were the same as those of 5-HT-IR neurons.TPH-IR/5-HT-IR in the brain and ventral nerve cord of Neanthes japonica had the same expression pattern,and TPH and 5-HT co-localized in the same position.It indicated that the specific TPH was responsible for the central serotonin synthesis in the central nervous system of Neanthes japonica.Therefore,TPH and 5-HT could be as the novel mutual corroboration marker to detect serotonergic neurons.Then,the full-length cDNA sequence of TPH in the central nervous system of Neanthes japonica was cloned by RACE technology,which was 1 778 bp and encodes predicted protein of 463 amino acid residues.Through phylogenetic analysis,it is proved that the existence of neural TPH at DNA/RNA level,so that the differentiation nodes of tph and pah gene in the AAAHs can be advanced fromArthropod to Annelida.2.The mouse anti-PAH polyclonal antibody was used to stained the paraffin sections of Neanthes japonica,and the results revealed that the antibody labeled many tadpole-like neurons in the whole brain,and the central neuropil region of midbrain and hindbrain is also covered with varicose-like nerve fibers.It corrected the conclusion that rabbit anti-PAH polyclonal antibody labeled the brain.The results also overturned previously hypothesis that the phenylalanine hydroxylase only converted phenylalanine to tyrosine in the cells without the need to secrete to the distal end of the cell.The distribution of PAH in the whole brain indicated that PAH can catalyze the conversion of phenylalanine to tyrosine in neuronal cells,but can also secrete to the distal,mainly in midbrain and hindbrain.The immunohistochemical staining of PAH indicated that PAH and TPH have been differentiated into independent genes in the lower organism Neanthes japonica,and have their expression products separately,which provide the evidence that the differentiation node of tph and pah genes may locate in Annelida.3.The results displayed that TH was distributed in the whole brain,and its location was similar to that of TPH and 5-HT,but not the same as PAH.According to the opinion that TH is a good marker of dopaminergic neuron cells,it can be seen that dopaminergic neurons can be labeled in the whole brain using the rabbit anti-TH polyclonal antibody.However,the strong positive immunoreactivity of dopaminergic neurons was relatively shorter,while the weak positive reaction was more than that of dopaminergic neurons.Dopaminergic neurons are mainly distributed in the dorsal side,most of the somata without synapse,only small number of somata have synapse with a short projection to the central neuropil region,which indicated that tyrosine is mainly hydroxylated in dopaminergic neurons by TH,only a small amount of enzyme was transported to the nearby and in the distance used for the hydroxylation.Furthermore,it was proved that the three aromatic amino acid hydroxylases in Neanthes japonica were independent of each other,which provided the evidences for the analysis of evolutionary node of tph,pah and th genes.4.The rabbit anti-D?H and rabbit anti-PNMT polyclonal antibodies were usedto stain the paraffin sections of Neanthes japonica by SP immunohistochemical method.The results showed that D?H had more groups of positive cells which distributed in the forebrain and less in midbrain and hindbrain,the soma in the midbrain and hindbrain was larger than that in the forebrain.However,the number of cell groups stained by anti-PNMT was relatively small.D?H is mainly located in the cell membrane of soma,only a small number of cells were full of D?H.The cellular distribution pattern of D?H was consistent with the existence of two types of higher biological cells in the state of dissolution and membrane binding state.However,PNMT is mainly located in the cytoplasm of soma,no distribution in the nucleus of the cells.Immunohistochemical staining of D?H and PNMT showed that norepinephrine and epinephrine located in the low invertebrate animal brain,and they played the regulatory role in different area of the brain.5.In summary,the monoamine neurotransmitters including 5-HT and catecholamines(dopamine,norepinephrine,epinephrine)are independent of each other in the central nervous system of Nereis Neanthes japonica.The synthesis of these monoamine neurotransmitters in vivo is closely related to the presence of three aromatic amino acid hydroxylases.