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

The role of nitric oxide ((NO)-N-.) 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)-N-. in vitro. Nation and o-phenylenediamine were used to modify the surface of carbon fiber microelectrodes (8 mu m diameter; approximate to 100 mu m tip length). Coating with Nafion was done at 170 degrees C and the o-phenylenediamine solution was electropolimerized on the carbon surface. (NO)-N-. peak potential (+0.78 +/- 0.03 V versus Ag/AgCl) was determined by square wave voltammetry with (NO)-N-. 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/mu M; n = 6) and linearity to (NO)-N-. 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)-N-. 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)-N-. dynamics in brain slices (in vitro) and possibly in whole brain (in vivo) recordings. (c) 2004 Elsevier B.V. All rights reserved.