In vivo real-time measurement of nitric oxide in anesthetized rat brain

During the last two decades nitric oxide (center dot NO) gas has emerged as a novel and ubiquitous intercellular modulator of cell functions. In the brain, center dot NO is implicated in mechanisms of synaptic plasticity but it is also involved in cell death pathways underlying several neurological diseases. Because of its hydrophobicity, small size, and rapid diffusion properties,the rate and pattern of center dot NO concentration changes are critical determinants for the understanding of its diverse actions in the brain. center dot NO measurement in vivo has been a challenging task due to its low concentration, short half-life, and high reactivity with other biological molecules, such as superoxide radical, thiols, and heme proteins. Electrochemical methods are versatile approaches for detecting and monitoring various neurotransmitters. When associated with microelectrodes inserted into the brain they provide high temporal and spatial resolution, allowing measurements of neurochemicals in physiological environments in a real-time fashion. To date, electrochemical detection of center dot NO is the only available technique that provides a high sensitivity, low detection limit, selectivity, and fast response to measure the concentration dynamics of center dot NO in vivo. We have used carbon fiber microelectrodes coated with two layers of Nafion and o-phenylenediamine to monitor the rate and pattern of center dot NO change in the rat brain in vivo. The analytical performance of microelectrodes was assessed in terms of sensitivity, detection limit, and selectivity ratios against major interferents: ascorbate, dopamine, noradrenaline, serotonin, and nitrite. For the in vivo recording experiments, we used a microelectrode/micropipette array inserted into the brain using a stereotaxic frame. The characterization of in vivo signals was assessed by electrochemical and pharmacological verification. Results support our experimental conditions that the measured oxidation current reflects variations in the center dot NO concentration in brain extracellular space. We report results from recordings in hippocampus and striatum upon stimulation of N-methyl-D-aspartate-subtype glutamate receptors. Moreover, the kinetics of center dot NO disappearance in vivo following pressure ejection of a center dot NO solution is also addressed.