Ana Ledo

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.

Neurovascular and neurometabolic derailment in aging and Alzheimer's disease

The functional and structural integrity of the brain requires local adjustment of blood flow and regulated delivery of metabolic substrates to meet the metabolic demands imposed by neuronal activation. This process—neurovascular coupling—and ensued alterations of glucose and oxygen metabolism—neurometabolic coupling—are accomplished by concerted communication between neural and vascular cells. Evidence suggests that neuronal-derived nitric oxide (•NO) is a key player in both phenomena. Alterations in the mechanisms underlying the intimate communication between neural cells and vessels ultimately lead to neuronal dysfunction. Both neurovascular and neurometabolic coupling are perturbed during brain aging and in age-related neuropathologies in close association with cognitive decline. However, despite decades of intense investigation, many aspects remain poorly understood, such as the impact of these alterations. In this review, we address neurovascular and neurometabolic derailment in aging and Alzheimers disease (AD), discussing its significance in connection with •NO-related pathways.

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.

Dynamic and interacting profiles of *NO and O2 in rat hippocampal slices.

Nitric oxide (*NO) is a ubiquitous signaling molecule that participates in the neuromolecular phenomena associated with memory formation. In the hippocampus, neuronal *NO production is coupled to the activation of the NMDA-type of glutamate receptor. Although *NO-mediated signaling has been associated with soluble guanylate cyclase activation, cytochrome oxidase is also a target for this gaseous free radical, for which *NO competes with O(2). Here we show, for the first time in a model preserving tissue cytoarchitecture (rat hippocampal slices) and at a physiological O(2) concentration, that endogenous NMDA-evoked *NO production inhibits tissue O(2) consumption for submicromolar concentrations. The simultaneous real-time recordings reveal a direct correlation between the profiles of *NO and O(2) in the CA1 subregion of the hippocampal slice. These results, obtained in a system close to in vivo models, strongly support the current paradigm for O(2) and *NO interplay in the regulation of cellular respiration.

Neurovascular uncoupling in the triple transgenic model of Alzheimer's disease: Impaired cerebral blood flow response to neuronal-derived nitric oxide signaling

Abstract Nitric oxide (•NO)‐dependent pathways and cerebrovascular dysfunction have been shown to contribute to the cognitive decline and neurodegeneration observed in Alzheimers disease (AD) but whether they represent initial factors or later changes of the disease is still a matter of debate. In this work, we aimed at investigating whether and to what extent neuronal‐derived •NO signaling and related neurovascular coupling are impaired along aging in the hippocampus of the triple transgenic mouse model of Alzheimers Disease (3xTg‐AD). We performed a longitudinal study combining behavior studies, in vivo simultaneous measurements of •NO concentration gradients and cerebral blood flow (CBF), along with detection of NO synthase (NOS) and markers of nitroxidative stress. Our results revealed an impairment in the neurovascular coupling along aging in the 3xTg‐AD mice which preceded obvious cognitive decline. This impairment was characterized by diminished CBF changes in response to normal or even increased •NO signals and associated with markers of nitroxidative stress. The results suggest that impairment in neurovascular coupling is primarily due to cerebrovascular dysfunction, rather than due to dysfunctional •NO signaling from neurons to blood vessels. Overall, this work supports cerebrovascular dysfunction as a fundamental underlying process in AD pathology. Graphical abstract Figure. No caption available. HighlightsNeurovascular coupling and nitrergic signaling pathways are altered in Alzheimers.The dysfunction in neurovascular coupling does not relate to nitric oxide signaling.Neurovascular coupling impairment appears to be primarily of cerebrovascular origin.

Neurovascular-neuroenergetic coupling axis in the brain: master regulation by nitric oxide and consequences in aging and neurodegeneration

Abstract The strict energetic demands of the brain require that nutrient supply and usage be fine‐tuned in accordance with the specific temporal and spatial patterns of ever‐changing levels of neuronal activity. This is achieved by adjusting local cerebral blood flow (CBF) as a function of activity level – neurovascular coupling – and by changing how energy substrates are metabolized and shuttled amongst astrocytes and neurons – neuroenergetic coupling. Both activity‐dependent increase of CBF and O2 and glucose utilization by active neural cells are inextricably linked, establishing a functional metabolic axis in the brain, the neurovascular‐neuroenergetic coupling axis. This axis incorporates and links previously independent processes that need to be coordinated in the normal brain. We here review evidence supporting the role of neuronal‐derived nitric oxide (•NO) as the master regulator of this axis. Nitric oxide is produced in tight association with glutamatergic activation and, diffusing several cell diameters, may interact with different molecular targets within each cell type. Hemeproteins such as soluble guanylate cyclase, cytochrome c oxidase and hemoglobin, with which •NO reacts at relatively fast rates, are but a few of the key in determinants of the regulatory role of •NO in the neurovascular‐neuroenergetic coupling axis. Accordingly, critical literature supporting this concept is discussed. Moreover, in view of the controversy regarding the regulation of catabolism of different neural cells, we further discuss key aspects of the pathways through which •NO specifically up‐regulates glycolysis in astrocytes, supporting lactate shuttling to neurons for oxidative breakdown. From a biomedical viewpoint, derailment of neurovascular‐neuroenergetic axis is precociously linked to aberrant brain aging, cognitive impairment and neurodegeneration. Thus, we summarize current knowledge of how both neurovascular and neuroenergetic coupling are compromised in aging, traumatic brain injury, epilepsy and age‐associated neurodegenerative disorders such as Alzheimers disease and Parkinsons disease, suggesting that a shift in cellular redox balance may contribute to divert •NO bioactivity from regulation to dysfunction. Graphical abstract Figure. No Caption available.

Age-dependent changes in the glutamate-nitric oxide pathway in the hippocampus of the triple transgenic model of Alzheimer's disease: implications for neurometabolic regulation

Age-dependent changes in nitric oxide ((•)NO) concentration dynamics may play a significant role in both decaying synaptic and metabolic functions in Alzheimers disease (AD). This neuromodulator acts presynaptically to increase vesicle release and glutamatergic transmission and also regulates mitochondrial function. Under conditions of altered intracellular redox environment, (•)NO may react and produce reactive species such as peroxynitrite. Using the triple transgenic mouse model of AD (3xTgAD), we investigated age-dependent changes in the glutamate-(•)NO axis in the hippocampus. Direct measurement of (•)NO concentration dynamics revealed a significant increase in N-methyl-D-aspartate type receptor-evoked peak (•)NO in the 3xTgAD model at an early age. Aging produced a decrease in peak (•)NO accompanied by significant decrease in production and decay rates in the transgenic model. Evaluation of energy metabolism revealed age-dependent decrease in basal oxygen consumption rate, a general decrease in mitochondrial oxidative phosphorylation parameters, and loss in mitochondrial sparing capacity in both genotypes. Finally, we observed age-dependent increase in 3-nitrotyrosine residues in the hippocampus, consistent with a putative shift in (•)NO bioactivity toward oxidative chemistry associated with neurotoxicity.

Age-Associated Changes of Nitric Oxide Concentration Dynamics in the Central Nervous System of Fisher 344 Rats

The increase in life expectancy is accompanied by an increased risk of developing neurodegenerative disorders and age is the most relevant risk factor for the appearance of cognitive decline. While decreased neuronal count has been proposed to be a major contributing factor to the appearance of age-associated cognitive decline, it appears to be insufficient to fully account for the decay in mental function in aged individuals. Nitric oxide (•NO) is a ubiquitous signaling molecule in the mammalian central nervous system. Closely linked to the activation of glutamatergic transmission in several structures of the brain, neuron-derived •NO can act as a neuromodulator in synaptic plasticity but has also been linked to neuronal toxicity and degenerative processes. Many studies have proposed that changes in the glutamate-•NO signaling pathway may be implicated in age-dependent cognitive decline and that the exact effect of such changes may be region specific. Due to its peculiar physical–chemical properties, namely hydrophobicity, small size, and rapid diffusion properties, the rate and pattern of •NO concentration changes are critical determinants for the understanding of its bioactivity in the brain. Here we show a detailed study of how •NO concentration dynamics change in the different regions of the brain of Fisher 344 rats (F344) during aging. Using microelectrodes inserted into the living brain of anesthetized F344 rats, we show here that glutamate-induced •NO concentration dynamics decrease in the hippocampus, striatum, and cerebral cortex as animals age. performance in behavior testing of short-term and spatial memory, suggesting that the impairment in the glutamate:nNOS pathway represents a functional critical event in cognitive decline during aging.

Neurometabolic and Electrophysiological Changes During Cortical Spreading Depolarization: Multimodal Approach Based on a Lactate-Glucose Dual Microbiosensor Arrays

Spreading depolarization (SD) is a slow propagating wave of strong depolarization of neural cells, implicated in several neuropathological conditions. The breakdown of brain homeostasis promotes significant hemodynamic and metabolic alterations, which impacts on neuronal function. In this work we aimed to develop an innovative multimodal approach, encompassing metabolic, electric and hemodynamic measurements, tailored but not limited to study SD. This was based on a novel dual-biosensor based on microelectrode arrays designed to simultaneously monitor lactate and glucose fluctuations and ongoing neuronal activity with high spatial and temporal resolution. In vitro evaluation of dual lactate-glucose microbiosensor revealed an extended linear range, high sensitivity and selectivity, fast response time and low oxygen-, temperature- and pH- dependencies. In anesthetized rats, we measured with the same array a significant drop in glucose concentration matched to a rise in lactate and concurrently with pronounced changes in the spectral profile of LFP-related currents during episodes of mechanically-evoked SD. This occurred along with the stereotypical hemodynamic response of the SD wave. Overall, this multimodal approach successfully demonstrates the capability to monitor metabolic alterations and ongoing electrical activity, thus contributing to a better understanding of the metabolic changes occurring in the brain following SD.

Combined in vivo Amperometric Oximetry and Electrophysiology in a Single Sensor – a Tool for Epilepsy Research

Seizures are paroxysmal events in which increased neuronal activity is accompanied by an increase in localized energetic demand. The ability to simultaneously record electrical and chemical events using a single sensor poses a promising approach to identify seizure onset zones in the brain. In the present work, we used ceramic-based platinum microelectrode arrays (MEAs) to perform high-frequency amperometric recording of local pO2 and local field potential (LFP)-related currents during seizures in the hippocampus of chronically implanted freely moving rats. Resting levels of O2 in the rodent brain varied between 6.6 ± 0.7 μM in the dentate gyrus (DG) region of the hippocampus and 22.1 ± 4.9 μM in the cerebral cortex. We also observed an expected increase in hippocampal pO2 (15 ± 4% from baseline) in response to tail pinch stress paradigm. Finally, induction of status epilepticus by intrahippocampal injection of pilocarpine induced biphasic changes in pO2 in the hippocampus. The initial dip at seizure onset (ΔO2 = -4.5 ± 0.7 μM) was followed by a prolonged hyperoxygenation phase (ΔO2 = +10.4 ± 2.9 μM). By acquiring the amperometry signal with a high sampling rate of 100 Hz we decomposed the raw signal in an oximetry recording (<1 Hz) and LFP recording (>1 Hz), demonstrating that each individual Pt site can simultaneously report changes in local pO2 and LFP-related currents during pilocarpine-induced seizure activity. This has high potential for translation into the clinical setting supported on intracranial grid or strip electrodes.