Production of Nitric Oxide within the Aplysia californica Nervous System

Abstract

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.

A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

“…Mass spectrometric analyses of DAFͲ2 has been described by others previously [22,41], but to the best of our knowledge this is the first time LCͲMS/MS has been used to analyze the fragmentation pattern of DAFͲFM and DAFͲFMͲT, and to confirm DAFͲFMͲT production in cells.…”

Molluscan cells in culture: primary cell cultures and cell lines

“…The following are examples of the main types of research conducted using cultured neuronal cells. (1) identification and functional characterization of neurotrophic factors regulating neurite outgrowth and regeneration (Spence et al 1998; Goldberg 1998; Munno et al 2000; Dmetrichuk et al 2008; Milanese et al 2009; Nejatbakhsh et al 2011 and others ), (2) growth cone motility and development (Spencer et al 1998; Dmetrichuk et al 2008; Suter 2011), (3) neurite-soma and soma-soma synaptogenesis (Munno et al 2000; Hu et al 2004; Onizuka et al 2012) and (4) neuronal receptors and cell-to-cell neurotransmitter signaling (White and Laczmarek 1997; Mapara et al 2008; Giachello et al 2010; Ye et al 2010). …”

Small-Volume Analysis of Cell–Cell Signaling Molecules in the Brain

“…The analytical techniques enabling NO detection at single-cell levels include fluorescence microscopy, CE with LIF detection, and electrochemistry (Ye et al, 2008). Using fluorescence confocal microscopy, CE, MS, and pharmacological tools, we verified the NO production in specific identified A. californica neurons (Ye et al, 2010). NO-selective amperometry has been used for dynamic measurements of NO in injured leeches, demonstrating that NO production is regulated through the endocannabinoid system (Arafah et al, 2013).…”

“…Various fluorescent NO indicators (Ye et al, 2010), electrochemical sensors (Arafah et al, 2013), or reporters of NO synthase activity (Ye et al, 2008;Potgieter et al, 2010) have been used to track NO in defined neuronal populations and regulatory pathways. The analytical techniques enabling NO detection at single-cell levels include fluorescence microscopy, CE with LIF detection, and electrochemistry (Ye et al, 2008).…”