Ricardo M. Santos

In Vivo Real‐Time Measurement of Nitric Oxide in Anesthetized Rat Brain

During the last two decades nitric oxide (.NO) gas has emerged as a novel and ubiquitous intercellular modulator of cell functions. In the brain, .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 .NO concentration changes are critical determinants for the understanding of its diverse actions in the brain. .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 .NO is the only available technique that provides a high sensitivity, low detection limit, selectivity, and fast response to measure the concentration dynamics of .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 .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 .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 .NO disappearance in vivo following pressure ejection of a .NO solution is also addressed.

Biomimetic sensor based on hemin/carbon nanotubes/chitosan modified microelectrode for nitric oxide measurement in the brain

A novel biomimetic microsensor for measuring nitric oxide (NO) in the brain in vivo was developed. The sensor consists of hemin and functionalized multi-wall carbon nanotubes covalently attached to chitosan via the carbodiimide crosslinker EDC followed by chitosan electrodeposition on the surface of carbon fiber microelectrodes. Cyclic voltammetry supported direct electron transfer from the Fe(III)/Fe(II) couple of hemin to the carbon surface at -0.370 V and -0.305 V vs. Ag/AgCl for cathodic and anodic peaks, respectively. Square wave voltammetry revealed a NO reduction peak at -0.762 V vs. Ag/AgCl that increased linearly with NO concentration between 0.25 and 1 μM. The average sensitivity of the microsensors was 1.72 nA/μM and the limit of detection was 25 nM. Oxygen and hydrogen peroxide reduction peaks were observed at -0.269 V and -0.332 V vs. Ag/AgCl, respectively and no response was observed for other relevant interferents, namely ascorbate, nitrite and dopamine. The microsensor was successfully applied to the measurement of exogenously applied NO in the rat brain in vivo.

A comparative study of carbon fiber-based microelectrodes for the measurement of nitric oxide in brain tissue.

The measurement of Nitric oxide (NO) in real-time has been a major concern due to the involvement of this ubiquitous free radical modulator in several physiological and pathological pathways in tissues. Here we performed a study aiming at evaluating different types of carbon fibers, namely Textron, Amoco, Courtaulds and carbon nanotubes (University of Kentucky) covered with Nafion/o-phenylenediamine (o-PD) for NO measurement in terms of sensitivity, LOD, response time and selectivity against major potential interferents in the brain (ascorbate, nitrite and dopamine). The results indicate that, as compared with the other carbon fibers and nanotubes, Textron carbon fiber microelectrodes coated with two layers of Nafion and o-PD exhibited better characteristics for NO measurement as they are highly selective against ascorbate (>30,000:1), nitrite (>2000:1) and dopamine (>80:1). These coated Textron microelectrodes showed an average sensitivity of 341+/-120pA/microM and a detection limit of 16+/-11nM. The better performance of the Textron fibers is likely related to a stronger adhesion or more uniform coating of the Nafion and o-PD polymers to the fiber surface. In addition, the background current of the Textron carbon fibers is low, contributing to the excellent signal-to-noise for detection of NO.

Neurovascular coupling in hippocampus is mediated via diffusion by neuronal-derived nitric oxide

The coupling between neuronal activity and cerebral blood flow (CBF) is essential for normal brain function. The mechanisms behind this neurovascular coupling process remain elusive, mainly because of difficulties in probing dynamically the functional and coordinated interaction between neurons and the vasculature in vivo. Direct and simultaneous measurements of nitric oxide (NO) dynamics and CBF changes in hippocampus in vivo support the notion that during glutamatergic activation nNOS-derived NO induces a time-, space-, and amplitude-coupled increase in the local CBF, later followed by a transient increase in local O2 tension. These events are dependent on the activation of the NMDA-glutamate receptor and nNOS, without a significant contribution of endothelial-derived NO or astrocyte-neuron signaling pathways. Upon diffusion of NO from active neurons, the vascular response encompasses the activation of soluble guanylate cyclase. Hence, in the hippocampus, neurovascular coupling is mediated by nNOS-derived NO via a diffusional connection between active glutamatergic neurons and blood vessels.

Nitric oxide signaling in the brain: translation of dynamics into respiration control and neurovascular coupling

The understanding of the unorthodox actions of neuronal‐derived nitric oxide (•NO) in the brain has been constrained by uncertainties regarding its quantitative profile of change in time and space. As a diffusible intercellular messenger, conveying information associated with its concentration dynamics, both the synthesis via glutamate stimulus and inactivation pathways determine the profile of •NO concentration change. In vivo studies, encompassing the real‐time measurement of •NO concentration dynamics have allowed us to gain quantitative insights into the mechanisms inherent to •NO‐mediated signaling pathways. It has been of particular interest to study the diffusion properties and half‐life, the interplay between •NO and O2 and the ensuing functional consequences for regulation of O2 consumption, the role of vasculature in shaping •NO signals in vivo, and the mechanisms that are responsible for •NO to achieve the coupling between glutamatergic neuronal activation and local microcirculation.

Evidence for a pathway that facilitates nitric oxide diffusion in the brain

Nitric oxide (()NO) is a diffusible messenger that conveys information based on its concentration dynamics, which is dictated by the interplay between its synthesis, inactivation and diffusion. Here, we characterized ()NO diffusion in the rat brain in vivo. By direct sub-second measurement of ()NO, we determined the diffusion coefficient of ()NO in the rat brain cortex. The value of 2.2×10(-5)cm(2)/s obtained in vivo was only 14% lower than that obtained in agarose gel (used to evaluate ()NO free diffusion). These results reinforce the view of ()NO as a fast diffusing messenger but, noticeably, the data indicates that neither ()NO diffusion through the brain extracellular space nor homogeneous diffusion in the tissue through brain cells can account for the similarity between ()NO free diffusion coefficient and that obtained in the brain. Overall, the results support that ()NO diffusion in brain tissue is heterogeneous, pointing to the existence of a pathway that facilitates ()NO diffusion, such as cell membranes and other hydrophobic structures.

The pattern of glutamate-induced nitric oxide dynamics in vivo and its correlation with nNOS expression in rat hippocampus, cerebral cortex and striatum

Nitric oxide (NO) is a diffusible intercellular messenger, acting via volume signaling in the brain and, therefore, the knowledge of its temporal dynamics is determinant to the understanding of its neurobiological role. However, such an analysis in vivo is challenging and indirect or static approaches are mostly used to infer NO bioactivity. In the present work we measured the glutamate-dependent NO temporal dynamics in vivo in the hippocampus (CA1, CA3 and DG subregions), cerebral cortex and striatum, using NO selective microelectrodes. Concurrently, the immunolocalization of nNOS was evaluated in each region. A transitory increase in NO levels occurred at higher amplitudes in the striatum and hippocampus relatively to the cortex. In the hippocampus, subtle differences in the profiles of NO signals were observed along the trisynaptic loop, with CA1 exhibiting the largest signals. The topography of NO temporal dynamics did not fully overlap with the pattern of the density of nNOS expression, suggesting that, complementary to the distribution of nNOS, the local regulation of NO synthesis as well as the decay pathways critically determine the effective NO concentration sensed by a target within the diffusional spread of this free radical. In sum, the rate and pattern of NO changes here shown, by incorporating regulatory mechanisms and processes that affect NO synthesis and decay, provide refined information critical for the understanding of NO multiple actions in the brain.

Nitric Oxide Inactivation Mechanisms in the Brain: Role in Bioenergetics and Neurodegeneration

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.

Brain nitric oxide inactivation is governed by the vasculature.

The mechanisms underlying nitric oxide ((•)NO) synthesis and inactivation in the brain are essential determinants of (•)NO neuroactivity. Although (•)NO production is well characterized, the pathways of inactivation in vivo remain largely unknown. Here, we characterize the kinetics and the major mechanism of (•)NO inactivation in the rat brain cortex and hippocampus in vivo by measuring locally applied (•)NO with carbon-fiber microelectrodes (CFMs) and ceramic-based microelectrode arrays (MEAs). An apparent first-order clearance was observed in both brain regions, with decay rate constants (k) of (•)NO signals of 0.67 to 0.84 per second, significantly higher than the k obtained in agarose gel (0.099 per second), used as a (•)NO diffusion-control medium. (•)NO half-life in vivo, estimated by mathematical modeling, was 0.42 to 0.75 s. Experiments using MEAs support that the (•)NO diffusion radius is heterogeneous and related to local metabolic activity and vascular density. After global ischemia, k decreased to control values of diffusion in gel, but during anoxia, k decreased only 21%. Additionally, k in brain slices was threefold to fivefold lower than that in vivo, and hemorrhagic shock induced a 53% decrease in k. Overall, the results support that (•)NO scavenging by circulating erythrocytes constitutes the major (•)NO-inactivation pathway in the brain.