PRODUCTION OF NITRIC OXIDE WITHIN THE APLYSIA CALIFORNICA NERVOUS SYSTEM.

Nitric oxide (NO), an intercellular signaling molecule, helps coordinate neuronal network activity. Here we examine NO generation in the Aplysia central nervous system using 4,5-diaminofluorescein diacetate (DAF-2 DA), a fluorescent reagent that forms 4,5-diaminofluorescein triazole (DAF-2T) upon reaction with NO. Recognizing that other fluorescence products are formed within the biochemically complex intracellular environment, we validate the observed fluorescence as being from DAF-2T; using both capillary electrophoresis and mass spectrometry we confirm that DAF-2T is formed from tissues and cells exposed to DAF-2 DA. We observe three distinct subcellular distributions of fluorescence in neurons exposed to DAF-2 DA. The first shows uniform fluorescence inside the cell, with these cells being among previously confirmed NOS-positive regions in the Aplysia cerebral ganglion. The second, seen inside buccal neurons, exhibits point sources of fluorescence, 1.5 ± 0.7 µm in diameter. Interestingly, the number of fluorescence puncta increases when the tissue is preincubated with the NOS substrate L-arginine, and they disappear when cells are preexposed to the NOS inhibitor L-NAME, demonstrating that the fluorescence is connected to NOS-dependent NO production. The third distribution type, seen in the R2 neuron, also exhibits fluorescent puncta, but only on the cell surface. Fluorescence is also observed in the terminals of cultured bag cell neurons loaded with DAF-2 DA. Surprisingly, fluorescence at the R2 surface and bag cell neuron terminals is not modulated by L-arginine or L-NAME, suggesting it has a source distinct from the buccal and cerebral ganglion DAF 2T-positive tissues.