Electrochemical measurement of endogenously produced nitric oxide in brain slices using Nafion/o-phenylenediamine modified carbon fiber microelectrodes

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 μm diameter; ≈100 μm 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/μM; 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.