I: Localization Of NgR Co-receptor Complex At Gap Junctions Between Pituicytes In Rats II: Morphological Study Of Pre-B(?)tzinger Complex-the Rhythmogenesis Center In Mediating Respiratory Neuroplasticity In Rats

Part I: The leading problem of neuroscience is thought to be the loss of regenerate capacity in the CNS after trauma. In the last two decades, the recognization of the substantial axon growth inhibitory factors in the adult CNS suggested that myelin proteins of oligodendrocyte might be the main source of these inhibitory factors and the mechanism underlying this effect drew vigous researches in this field.NgR was first identified as a neuronal receptor for an oligodendrocyte-derived axon growth inhibitor Nogo-A, targeting at its Nogo-66 domain. It was recently reported that NgR had been found in the astrocytes as well as in the neurons. Our previous study revealed that NgR could locate on the gap junctions between pituicytes. This suggested that NgR might have new function of regulating the activities of glial contact. Because NgR do not have intracellular segment, the co-receptors such as LINGO-1/p75/TROY are needed for NgR to work. To investigate which one or more co-receptors play a role at gap junctions in posterior pituitary, we double-labelled LINGO-1/p75/TROY and marker of gap junction-Cx43. Through the observation under light and electronic microscope, we found:1. LINGO-1/p75/TROY-ir was scattered in the posterior pituitary of rat with a considerable amount and co-localized with Cx43 numerously;2. Under electron microscope, LINGO-1/p75/TROY could be seen in the cytoplasma of pituicytes. But we did not found them distributed on the gap junction.3. In this study, we found that generally accepted marker of pituicytes GFAP only presented in a few amount of them and another kind of marker of astrocyte-like cells S100βcan mark more pituicytes under light microscopic level. Under electron microscope, GFAP only represented in one subtype of pituicyte but S100βin both.Above all, we speculate that there might be one or more new co-receptors function at gap junctions between pituicytes instead of those already known. Moreover, S100βmight be a better marker of pituicytes.Part II: Respiratory neuroplasticity represents a respiratory persistent change based on experience and adaptation to demands under physiological or pathological conditions. Failure of these mechanisms may affect stable airway tone and potentially lead to sleep disorders, such as obstructive sleep apnoea and sudden infant death syndrome. Long-term facilitation (LTF), a most frequently studied model of respiratory neuroplasticity that can be induced by episodic hypoxia to produce a long-lasting enhancement of phrenic motor output, is proposed to be mediated through 5-HT_2A receptor (5-HT_2AR) mechanism. Howerer, a body of studies referring to respiratory LTF have been focused on the locus of phrenic nucleus in the cervical spinal cord. Whether the pre-B(o|¨)tzinger complex (pre-B(o|¨)tC), a proposed kernel of respiratory rhythmogenesis, contributes to the activity of respiratory plasticity remains little known so far. We established a hypobaric rat model that was induced by chronic intermittent hypoxia (CIH) to generate longlasting augmented phrenic LTF. Using this established LTF model, the present study wished to investigate the morphological phenotypes of 5-HT/5-HT_2AR system and its downstream intracellular signalings, including phospho (P)-PKCθ/P-PKC substrate, and P-CaMKII/P-CREB in the pre-B(o|¨)tC between the control and CIH rats, in combination with neurokinin 1 receptor (NK1R) as a marker of the pre-B(o|¨)tC. We found that 5-HT and 5-HT_2AR were highly expressed in the pre-B(o|¨)tC in CIH animals compared with the control. Of particularly interesting was the transmembrane distribution of 5-HT_2AR in the pre-B(o|¨)tC in CIH rats. 5-HT_2AR immunoreactivity was mainly associated with the inner surface of the plasma membrane in control rats. However, in CIH rats, 5-HT_2AR immunoreactivity was densely localized not only along the inner surface of the plasma membrane, but also along the outer surface or on the plasma membrane of pre-B(o|¨)tC neurons. In the invaginations of the plasma membrane, where receptor endocytosis or exocytosis might occur, 5-HT_2AR was also detectable. The 5-HT_2AR transmembrane distribution implyed CIH-induced high activation of 5-HT/5-HT_2AR system in the pre-B(o|¨)tC. Accordingly, P-PKCθ/P-PKC substrate and P-CaMKII/ P-CREB were intensely expressed in the pre-B(o|¨)tC in CIH animals in contrast to the control, which was evident with Western blot probing proteins of the two groups. Of notable was the close association of P-PKCθand P-CaMKII with postsynaptic density, implicating the postsynaptic mechanisms by which the pre-B(o|¨)tC neurons might exert their effects to mediate respiratory neuroplasticity.5-HT_2AR is a G-protein coupled receptor, and its function relies on second messengers'activation. PKC cascade is a known pathway triggered by 5-HT_2AR via an increase in IP3 and diacylglycerol, of them IP3 then activate CaMKII. Indeed, the present findings provided morphological evidence of existing 5-HT→5-HT_2AR→P-PKCθand 5-HT→5-HT_2AR→CaMKII→CREB pathways in the pre-B(o|¨)tC in control rats. Particularly, the pathways were highly activated followed with CIH pretreatment. The present study establishes the cellular neurochemical basis for understanding functional respiratory neuroplasticity in the pre-B(o|¨)tC.