This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mucociliary activity is an important clearance mechanism in the respiratory system of air breathing vertebrates. An in depth knowledge of the regulation of mucociliary activity may assist in the development of new treatments for various clinical conditions such as asthma, chronic sinusitis, cystic fibrosis and bronchitis which are associated with impaired mucociliary activity. Increased mucociliary activity during these conditions is beneficial in the sense that pharmacological or physiotherapeutic manipulation of this reflex may be of therapeutic value. The administration of substance P (SP) and acetylcholine (ACh) as well as electrical stimulation of the palatine nerve were shown to increase mucociliary activity whereas dopamine and serotonin were shown to inhibit mucocitiary activity. Hernandez, Berrios and Chinapen (2003) found: SP-labeled cell bodies in the trigeminal ganglion from which the palatine nerve emerges; SP-labeled fibers innervating the palate; and SP-labeled fibers innervating the blood vessels of the palate. However, it remains to be established if SP-labeled cell bodies are the cells of origin of the palatine nerve? Once this question is addressed, the localization of SP, NK-1 receptors in this pathway will be determined and its interaction with SP fibers will be studied. Since ACh also regulates mucociliary activity it is necessary to determine whether it is localized in this pathway. Once this is established the identification and localization of the ACh muscarinic receptor subtype in the palate and its interaction with ChAT fibers will also be addressed. Therefore, the long term goal of this study is to elucidate the neuronal pathways mediating mucociliary activity using the palate of the frog Rana pipiens as an animal model. The results of this experiment will be compared with future studies in mammals. The experiments of this proposal are: I. Identification of SP in the pathway mediating mucociliary activity. - To determine whether the trigeminal ganglion contains the cells of origin of the palatine nerve. The retrograde tracer, Fluoro Gold and the anterograde tracer, Biotinylated Dextran Amine will be used to identify the cells and terminals respectively. It is hypothesized that the trigeminal ganglion contains the cells of origin of the palatine nerve. - To determine whether the cells of origin of the palatine nerve (identified in specific aim la) contain SP. This will be accomplished by double-labeling procedures combining retrograde and/or anterograde labeled sections with the immunofluorescent labeling for SP. It is hypothesized that the SP-labeled neurons of the trigeminal ganglion are the cells of origin of the palatine nerve. - To determine if BDA anterograde labeled nerve terminals contain SP and if so whether they synapse with mucus and epithelia cells of the palate using immunoelectron microscopy. It is hypothesized that there are SP-containing terminals snapping on the goblet and ciliated epithelial cells of the palate. - To localize SP, NK-1 receptors in the palate and medulla and demonstrate its interaction with SP-labeled BDA containing terminals. This will be accomplished with a triple labeling procedure combining BDA labeled sections with the immunocytochemical labeling for SP, NK-lreceptor and SP. It is hypothesized that SPlabeled BDA containing terminals interact with SP, NK-1 receptors in the palate and medulla. II. Identification of acetylcholine in the pathway mediating mucociliary activity. - To localize choline acetyl transferase (ChAT)-containing cell bodies in the trigeminal ganglion and in the fibers that innervate the palate. This will be accomplished with the immunocytochemical method to detect CHAT; the enzyme that catalyzes the synthesis of acetylcholine. It is hypothesized that there are ChAT containing cell bodies in the trigeminal ganglion and ChAT containing fibers in the palate of Rana pipiens. - To determine whether SP and ChAT -fibers in the palate are labeled with BDA injected into the trigeminal ganglion. This will be accomplished by a triple-labeling procedure combining anterograde labeled sections with the immunocytochemical labeling for SP and CHAT. It is hypothesized that SP, ChAT and/or BDA are colocalized in the same neurons. - To localize the ACh muscarinic receptor subtype (s) in the palate and demonstrate its interaction with CHATlabeled BDA fibers identified in the palate. This will be accomplished with a triple labeling procedure combining BDA labeled sections with the immunocytochemical labeling for ACh muscarinic receptor and CHAT. It is hypothesized that ChAT-labeled BDA containing terminals interact with ACh muscarinic receptor(s) in the palate and medulla.
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