The Expression Of Substance P And Substance P Receptor In Development Of Mice Brain

Substance P expression is confined to central nervous system (CNS) and the peripheral nervous system (PNS). Today it is fully recognized that substance P is released from both the central and peripheral endings of primary afferent neurons and functions as a neurotransmitter.Mammalian substance P derives from the preprotachykinin- A (PPT-A) gene, The PPT-A gene also encodes for other tachykinins, including neurokinin A, neurokinin B. The three tachykinins have their receptors such as NK₁ receptor, NK₂ receptor and NK₃ receptor. NK₁ receptor has the highest affinity with substance P, also be called as substance P receptor. When substance P binding NK₁ receptor, NK₁ receptor internalization, activate phosphatidolipaseC and hydrolyze phosphinositides, generate two second message IP3 and DAG to induce biological effect.Finished experimentation show substance P is already expression in embryo brain prenatal, but also have no idea with its role in the development of central nerves system. There also have no report about the expression of substance P receptor in the development of central nerves system. So it is very necessary to investigate the expression and localization of substance P and substance P receptor, can provide the morphology proof in research about substance P'function in the development of central nerves system.Materials and Methods1. Animals and histological section preparationAnimals: El l(Embryonic mice at 1 Id), E13, E15, E17, E19 and PO (new born) Sections: All of mice brians were cut in cryostat coronal sections.2. ImmunohistochemistryFor immunocytochemistry, sections were incubated with polyclonal rabbit-anti-SP or SPR antibody, biotinylated goat-anti-rabbit secondary antibody and ABC complex, respectively.3. Western blottingEll, E13, E15, E17, E19 and PO mice brians were removed, respectively. After homogenation, they were taken to PAGE, and transferred to nitrocellulose membrane. Membranes were incubated with polyclonal anti-SP or SPR antibody, biotinylated goat-anti-rabbit secondary antibody and ABC complex, respectively.Resultsl.SP and SPR expression in the development of embryo mice brainOn Ell, SP-immunoreactive neurons were detected in the marginal zone of caudal epithalamus neuronepithelium, while positive-fibers were not seen. Some SPR-immunoreactive cells were seen in the ventral hypothalamus, pons and cingulate cortex.On El 3, SP-immunoreactive neurons were found in the striatum, globus pallidus and septal area. SPR-immunoreactive neurons were seen in the medullary raphe, olfactory brain, hypothalamus. Positive neural fibers were seen in the ventral tegmental nucleus (VTG).On El5, the dense stained SP-immunoreactive neurons existed at olfactory brain, septal area, piriform cortex and diagonal band, and also appears at striatum, globus pallidus and nucleus accumbens. The distribution of SPR-immunoreactive neurons was found to be similar to the distribution of SP. SPR-immunoreactive neurons were found in the olfactory brain, septal area, piriform cortex, hippocampus, amygdala and striatum,especially at medulla oblongata. The neurons were faintly stained.On El7, SP-immunoreactive cells beam was found from entopeduncular nucleus and bed nucleus of the stria terminalisto the striatum through the internal capsule and globus pallidus. Dense stained largeround or fusiform cells with distinct fibre projections were observed. SP-immunoreactive neurons also can be found in the hippocampus and gyrus dentatus, and SP-immunoreactive neurons terminus were found in the medial amygdaloid necleus. SPR-immunoreactive cells were also found in the same region such as striatum and globus pallidus. SPR-immunoreactive fibers were found in the amygdale, ventral thalamus, lateral hypothalamus and the raphe region of medulla oblongata such as nucleus raphes dorsalis, locus caeruleus, central gray and superior raphe nucleus.On El9, lots of SP-immunoreactive termini were observed in the entopeduncular nucleus, Forel region, thalamus, anterior colliculi, periaqueductal gray, reticular formation, inferior olivary nucleus, ventral tegmental nucleus and locus caeruleus. SP-immunoreactive cells were found in the epithalamus, ganglion habenulae, entopeduncular nucleus, central gray, dorsal tegmental nucleus and interpeduncular nucleus.On PO, SP-immunoreactive cells were found in the hypothalamus, entopeduncular nucleus, striatum, amygdale, reticular nucleus of raphe in medulla oblongata, nucleus raphes dorsalis and zona incerta, cells were densely stained and accompanied with lots of terminus. SPR-immunoreactive fibres appeared in the amygdala, striatum, nucleus accumbens, central gray and ventral tegmental nucleus. 2. Western blottingWe found SP began expressed at Ell and gradually increasing. Then in El5 and El7 increased significantly until birth, SPR began expressed at Ell and have stable expression until birth.Conclusions1. Substance p began expressed at neostriatum in the early embryo development, while substance P receptor expressed at raphe of medulla oblongata;2. In the midanaphase of embryo development SP and SPR were found in the same place such as striatum and raphe of medulla oblongata, but the shape of positive-cells was different.3. On early stage of developmental 1) SP expression can be found and gradually increased, at El5 and El7 the expression increased significantly until birth. The expression of SPR also can be found on Ell and maintained stable expression, before birth the expression increased.4. It suggested that SP may play a role in central nervous system genesis and mature mechanism.