title: 4,5-diaminofluoroscein imaging of nitric oxide synthesis in crayfish terminal ganglia.
abstract: We have analyzed the synthesis of nitric oxide in the terminal abdominal ganglion of the crayfish using the fluorescent probe 4,5-Diaminofluoroscein diacetate, DAF-2 DA. Following DAF-2 loading, ganglia showed cell-specific patterns of fluorescence in which the occurrence of strongly fluorescent cell bodies was highest in specific anterior, central, and posterior regions. We found that preincubation with the nitric oxide synthase (NOS) inhibitor L-NAME prevented much of the initial development of DAF-2 fluorescence, whereas the inactive isomer D-NAME had no effect. Washout of preincubated L-NAME caused increased cell-specific fluorescence due to endogenous NOS activity. Application of the NOS substrate L-arginine also resulted in an increase of DAF-2 fluorescence in a cell-specific manner. We bath applied the NO donor SNAP to increase exogenous NO levels which resulted in DAF-2 fluorescence increases in most cells. We therefore presume that the cell-specific pattern of DAF-2 fluorescence indicates the distribution of neurones actively synthesizing NO. The similarity between the DAF-2 staining pattern and previously published studies of NOS activity are discussed.
authors.affiliation: School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, United Kingdom.
authors.forename: Hansjürgen
authors.initials: H
authors.lastname: Schuppe
authors.forename: Matthew
authors.initials: M
authors.lastname: Cuttle
authors.forename: John E
authors.initials: JE
authors.lastname: Chad
authors.forename: Philip L
authors.initials: PL
authors.lastname: Newland
mesh_terms: D000818:Animals; D003400:Astacoidea; D005260:Female; D019793:Fluorescein / Q000032:analysis*; D005453:Fluorescence; D017950:Ganglia, Sensory / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000032:analysis* / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: C111205:4,5-diaminofluorescein; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D019793:Fluorescein; D019331:NG-Nitroarginine Methyl Ester