carbon fiber microelectrode ͉ NO diffusional spread ͉ hippocampus

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The role of nitric oxide ( • NO) as a regulatory diffusible molecule in the brain requires the evaluation of its concentration dynamics. In this work, we have developed microelectrodes suitable for real time electrochemical measurements of • NO in vitro. Nafion and o-phenylenediamine were used to modify the surface of carbon fiber microelectrodes (8 m diameter; ≈100 m tip length). Coating with Nafion was done at 170 • C and the o-phenylenediamine solution was electropolimerized on the carbon surface. • NO peak potential (+0.78 ± 0.03 V versus Ag/AgCl) was determined by square wave voltammetry with • NO solutions prepared from the-generating compound diethylenetriamine/nitric oxide (DETA/NO). Microelectrodes were calibrated by amperometry at a potential of +0.90 V versus Ag/AgCl. They showed good sensitivity (954 ± 217 pA/M; n = 6) and linearity to • NO in the concentration range of 100-1000 nM. They were also characterized in terms of detection limit (6 ± 2 nM, n = 4), response time at 50% (1 s), and selectivity against interferents, such as nitrite (780 ± 84:1, n = 6), ascorbic acid (750 ± 187:1, n = 6) or dopamine (18 ± 2:1, n = 6). Injections of 1 mM l-glutamate, 1 mM l-arginine, and 0.1 mM N-methyl-d-aspartate did not produce changes in background current. Finally, the microelectrodes were used to measure • NO concentration dynamics in rat hippocampal brain slices stimulated with l-glutamate and N-methyl-d-aspartate. Taken together, the data indicate that the microelectrodes exhibit the proper sensitivity and selectivity for studies of • NO dynamics in brain slices (in vitro) and possibly in whole brain (in vivo) recordings.

During the last decades nitric oxide (•NO) has emerged as a critical physiological signaling molecule in mammalian tissues, notably in the brain. •NO may modify the activity of regulatory proteins via direct reaction with the heme moiety, or indirectly, via S-nitrosylation of thiol groups or nitration of tyrosine residues. However, a conceptual understanding of how •NO bioactivity is carried out in biological systems is hampered by the lack of knowledge on its dynamics in vivo. Key questions still lacking concrete and definitive answers include those related with quantitative issues of its concentration dynamics and diffusion, summarized in the how much, how long, and how far trilogy. For instance, a major problem is the lack of knowledge of what constitutes a physiological •NO concentration and what constitutes a pathological one and how is •NO concentration regulated. The ambient •NO concentration reflects the balance between the rate of synthesis and the rate of breakdown. Much has been learnt about the mechanism of •NO synthesis, but the inactivation pathways of •NO has been almost completely ignored. We have recently addressed these issues in vivo on basis of microelectrode technology that allows a fine-tuned spatial and temporal measurement •NO concentration dynamics in the brain.