From synaptically localized to volume transmission by nitric oxide.

Nitric oxide (NO) functions widely as a transmitter/diffusible second messenger in the central nervous system, exerting physiological effects in target cells by binding to specialized guanylyl cyclase-coupled receptors, resulting in cGMP generation. Despite having many context-dependent physiological roles and being implicated in numerous disease states, there has been a lack of clarity about the ways that NO operates at the cellular and subcellular levels. Recently, several approaches have been used to try to gain a more concrete, quantitative understanding of this unique signalling pathway. These approaches have included analysing the kinetics of NO receptor function, real-time imaging of cellular NO signal transduction in target cells, and the use of ultrasensitive detector cells to record NO as it is being generated from native sources in brain tissue. The current picture is that, when formed in a synapse, NO is likely to act only very locally, probably mostly within the confines of that synapse, and to exist only in picomolar concentrations. Nevertheless, closely neighbouring synapses may also be within reach, raising the possibility of synaptic crosstalk. By engaging its enzyme-coupled receptors, the low NO concentrations are able to stimulate physiological (submicromolar) increases in cGMP concentration in an activity-dependent manner. When many NO-emitting neurones or synapses are active simultaneously in a tissue region, NO can act more like a volume transmitter to influence, and perhaps coordinate, the behaviour of cells within that region, irrespective of their identity and anatomical connectivity.

Nitric oxide targets oligodendrocytes and promotes their morphological differentiation.

In the central nervous system, nitric oxide (NO) transmits signals from one neurone to another, or from neurones to astrocytes or blood vessels, but the possibility of oligodendrocytes being physiological NO targets has been largely ignored. By exploiting immunocytochemistry for cGMP, the second messenger generated on activation of NO receptors, oligodendrocytes were found to respond to both exogenous and endogenous NO in cerebellar slices from rats aged 8 days to adulthood. Atrial natriuretic peptide, which acts on membrane-associated guanylyl cyclase-coupled receptors, also raised oligodendrocyte cGMP in cerebellar slices. The main endogenous source of NO accessing oligodendrocytes appeared to be the neuronal NO synthase isoform, which was active even under basal conditions and in a manner that was independent of glutamate receptors. Oligodendrocytes in brainstem slices were also shown to be potential NO targets. In contrast, in the optic nerve, oligodendrocyte cGMP was raised by natriuretic peptides but not NO. When cultures of cerebral cortex were continuously exposed to low NO concentrations (estimated as 40-90 pM), oligodendrocytes responded with a striking increase in arborization. This stimulation of oligodendrocyte growth could be replicated by low concentrations of 8-bromo-cGMP (maximum effect at 1 µM). It is concluded that oligodendrocytes are probably widespread targets for physiological NO (or natriuretic peptide) signals, with the resulting rise in cGMP serving to enhance their growth and maturation. NO might help coordinate the myelination of axons to the ongoing level of neuronal activity during development and could potentially contribute to adaptive changes in myelination in the adult.

A new small molecule inhibitor of soluble guanylate cyclase.

Soluble guanylate cyclase (sGC) is a haem containing enzyme that regulates cardiovascular homeostasis and multiple mechanisms in the central and peripheral nervous system. Commonly used inhibitors of sGC activity act through oxidation of the haem moiety, however they also bind haemoglobin and this limits their bioavailability for in vivo studies. We have discovered a new class of small molecule inhibitors of sGC and have characterised a compound designated D12 (compound 10) which binds to the catalytic domain of the enzyme with a KD of 11 µM in a SPR assay.

Lesional-targeting of neuroprotection to the inflammatory penumbra in experimental multiple sclerosis.

Progressive multiple sclerosis is associated with metabolic failure of the axon and excitotoxicity that leads to chronic neurodegeneration. Global sodium-channel blockade causes side effects that can limit its use for neuroprotection in multiple sclerosis. Through selective targeting of drugs to lesions we aimed to improve the potential therapeutic window for treatment. This was assessed in the relapsing-progressive experimental autoimmune encephalomyelitis ABH mouse model of multiple sclerosis using conventional sodium channel blockers and a novel central nervous system-excluded sodium channel blocker (CFM6104) that was synthesized with properties that selectively target the inflammatory penumbra in experimental autoimmune encephalomyelitis lesions. Carbamazepine and oxcarbazepine were not immunosuppressive in lymphocyte-driven autoimmunity, but slowed the accumulation of disability in experimental autoimmune encephalomyelitis when administered during periods of the inflammatory penumbra after active lesion formation, and was shown to limit the development of neurodegeneration during optic neuritis in myelin-specific T cell receptor transgenic mice. CFM6104 was shown to be a state-selective, sodium channel blocker and a fluorescent p-glycoprotein substrate that was traceable. This compound was >90% excluded from the central nervous system in normal mice, but entered the central nervous system during the inflammatory phase in experimental autoimmune encephalomyelitis mice. This occurs after the focal and selective downregulation of endothelial p-glycoprotein at the blood-brain barrier that occurs in both experimental autoimmune encephalomyelitis and multiple sclerosis lesions. CFM6104 significantly slowed down the accumulation of disability and nerve loss in experimental autoimmune encephalomyelitis. Therapeutic-targeting of drugs to lesions may reduce the potential side effect profile of neuroprotective agents that can influence neurotransmission. This class of agents inhibit microglial activity and neural sodium loading, which are both thought to contribute to progressive neurodegeneration in multiple sclerosis and possibly other neurodegenerative diseases.

Surface plasmon resonance using the catalytic domain of soluble guanylate cyclase allows the detection of enzyme activators.

Soluble Guanylate Cyclase (sGC) is the receptor for the signalling agent nitric oxide (NO) and catalyses the production of the second messenger cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). The enzyme is an attractive drug target for small molecules that act in the cardiovascular and pulmonary systems, and has also shown to be a potential target in neurological disorders. We have discovered that 5-(indazol-3-yl)-1,2,4-oxadiazoles activate the enzyme in the absence of added NO and shown they bind to the catalytic domain of the enzyme after development of a surface plasmon resonance assay that allows the biophysical detection of intrinsic binding of ligands to the full length sGC and to a construct of the catalytic domain.

Exquisite sensitivity to subsecond, picomolar nitric oxide transients conferred on cells by guanylyl cyclase-coupled receptors.

Nitric oxide (NO) functions as a diffusible transmitter in most tissues of the body and exerts its effects by binding to receptors harboring a guanylyl cyclase transduction domain, resulting in cGMP accumulation in target cells. Despite its widespread importance, very little is known about how this signaling pathway operates at physiological NO concentrations and in real time. To address these deficiencies, we have exploited the properties of a novel cGMP biosensor, named δ-FlincG, expressed in cells containing varying mixtures of NO-activated guanylyl cyclase and cGMP-hydrolyzing phosphodiesterase activity. Responsiveness to NO, signifying a physiologically relevant rise in cGMP to 30 nM or more, was seen at concentrations as low as 1 pM, making cells by far the most sensitive NO detectors yet encountered. Even cells coexpressing phosphodiesterase-5, a cGMP-activated isoform found in many NO target cells, responded to NO in concentrations as low as 10 pM. The dynamics of NO capture and signal transduction was revealed by administering timed puffs of NO from a local pipette. A puff lasting only 100 ms, giving a calculated peak intracellular NO concentration of 23 pM, was detectable. The results could be encapsulated in a quantitative model of cellular NO-cGMP signaling, which recapitulates the NO responsiveness reported previously from crude cGMP measurements on native cells, and which explains how NO is able to exert physiological effects at extremely low concentrations, when only a tiny proportion of its receptors would be occupied.

The Concise Guide to PHARMACOLOGY 2023/24: Catalytic receptors.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and nearly 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16180. Catalytic receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Picomolar nitric oxide signals from central neurons recorded using ultrasensitive detector cells.

Nitric oxide (NO) is a widespread signaling molecule with potentially multifarious actions of relevance to health and disease. A fundamental determinant of how it acts is its concentration, but there remains a lack of coherent information on the patterns of NO release from its sources, such as neurons or endothelial cells, in either normal or pathological conditions. We have used detector cells having the highest recorded NO sensitivity to monitor NO release from brain tissue quantitatively and in real time. Stimulation of NMDA receptors, which are coupled to activation of neuronal NO synthase, routinely generated NO signals from neurons in cerebellar slices. The average computed peak NO concentrations varied across the anatomical layers of the cerebellum, from 12 to 130 pm. The mean value found in the hippocampus was 200 pm. Much variation in the amplitudes recorded by individual detector cells was observed, this being attributable to their location at variable distances from the NO sources. From fits to the data, the NO concentrations at the source surfaces were 120 pm to 1.4 nm, and the underlying rates of NO generation were 36-350 nm/s, depending on area. Our measurements are 4-5 orders of magnitude lower than reported by some electrode recordings in cerebellum or hippocampus. In return, they establish coherence between the NO concentrations able to elicit physiological responses in target cells through guanylyl cyclase-linked NO receptors, the concentrations that neuronal NO synthase is predicted to generate locally, and the concentrations that neurons actually produce.

NOS1 mutations cause hypogonadotropic hypogonadism with sensory and cognitive deficits that can be reversed in infantile mice.

The nitric oxide (NO) signaling pathway in hypothalamic neurons plays a key role in the regulation of the secretion of gonadotropin-releasing hormone (GnRH), which is crucial for reproduction. We hypothesized that a disruption of neuronal NO synthase (NOS1) activity underlies some forms of hypogonadotropic hypogonadism. Whole-exome sequencing was performed on a cohort of 341 probands with congenital hypogonadotropic hypogonadism to identify ultrarare variants in NOS1. The activity of the identified NOS1 mutant proteins was assessed by their ability to promote nitrite and cGMP production in vitro. In addition, physiological and pharmacological characterization was carried out in a Nos1-deficient mouse model. We identified five heterozygous NOS1 loss-of-function mutations in six probands with congenital hypogonadotropic hypogonadism (2%), who displayed additional phenotypes including anosmia, hearing loss, and intellectual disability. NOS1 was found to be transiently expressed by GnRH neurons in the nose of both humans and mice, and Nos1 deficiency in mice resulted in dose-dependent defects in sexual maturation as well as in olfaction, hearing, and cognition. The pharmacological inhibition of NO production in postnatal mice revealed a critical time window during which Nos1 activity shaped minipuberty and sexual maturation. Inhaled NO treatment at minipuberty rescued both reproductive and behavioral phenotypes in Nos1-deficient mice. In summary, lack of NOS1 activity led to GnRH deficiency associated with sensory and intellectual comorbidities in humans and mice. NO treatment during minipuberty reversed deficits in sexual maturation, olfaction, and cognition in Nos1 mutant mice, suggesting a potential therapy for humans with NO deficiency.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Catalytic receptors.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15541. Catalytic receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Mechanisms of activity-dependent plasticity in cellular nitric oxide-cGMP signaling.

Cellular responsiveness to nitric oxide (NO) is shaped by past history of NO exposure. The mechanisms behind this plasticity were explored using rat platelets in vitro, specifically to determine the relative contributions made by desensitization of NO receptors, which couple to cGMP formation, and by phosphodiesterase-5 (PDE5), which is activated by cGMP and also hydrolyzes it. Repeated delivery of brief NO pulses (50 nM peak) at 1-min intervals resulted in a progressive loss of the associated cGMP responses, which was the combined consequence of receptor desensitization and PDE5 activation, with the former dominating. Delivery of pulses of differing amplitude showed that NO stimulated and desensitized receptors with similar potency (EC50 = 10-20 nM). PDE5 activation was highly sensitive to NO, with a single pulse peaking at 2 nM being sufficient to evoke a 50% loss of response to a subsequent near-maximal NO pulse. However, the activated state of the PDE subsided quickly after removal of NO, the half-time for recovery being 25 s. In contrast, receptor desensitization reverted much more slowly, the half-time being 16 min. Accordingly, with long (20-min) exposures, NO concentrations as low as 600 pM provoked significant desensitization. The results indicate that PDE5 activation and receptor desensitization subserve distinct short term and longer term roles as mediators of plasticity in NO-cGMP signaling. A kinetic model explicitly describing the complex interplay between NO concentration, cGMP synthesis, PDE5 activation, and the resulting cGMP accumulation successfully simulated the present and previous data.

Hyperpolarization-activated ion channels as targets for nitric oxide signalling in deep cerebellar nuclei.

Most biological effects of nitric oxide (NO) in the brain are mediated by guanylyl cyclase-coupled NO receptors, whose activation results in increased intracellular cGMP levels. Apart from protein kinase activation little is known about subsequent cGMP signal transduction. In optic nerve axons, hyperpolarization-activated cyclic nucleotide-modulated cation (HCN) channels, which bind cGMP or cAMP directly, were recently suggested to be a target. The aim here was to test this possibility more directly. Neurones of the rat deep cerebellar nuclei were selected for this purpose, their suitability being attested by immunocytochemistry showing that the principal neurones expressed guanylyl cyclase protein and that NO synthase-containing fibres were abundant in the neuropil. Using whole-cell voltage-clamp recording, HCN channels in the neurones were activated in response to isoprenaline and exogenous cAMP but only occasionally did they respond to NO, although exogenous cGMP was routinely effective. With the less invasive sharp microelectrode recording technique, however, exogenous NO modulated the channels reproducibly, as measured by the size of the HCN channel-mediated voltage sag following hyperpolarization. Moreover, NO also blunted the subsequent rebound depolarizing potentials, consistent with it increasing the hyperpolarization-activated current. Optimizing the whole-cell solution to improve the functioning of NO-activated guanylyl cyclase failed to restore NO sensitivity. Minimizing cellular dialysis by using the perforated-patch technique, however, was successful. The results provide evidence that HCN channels are potential downstream mediators of NO signalling in deep cerebellar nuclei neurones and suggest that the more general importance of this transduction pathway may have been overlooked previously because of unsuitable recording methods.

New insight into the functioning of nitric oxide-receptive guanylyl cyclase: physiological and pharmacological implications.

The cellular counterpart of the "soluble" guanylyl cyclase found in tissue homogenates over 30 years ago is now recognized as the physiological receptor for nitric oxide (NO). The ligand-binding site is a prosthetic haem group that, when occupied by NO, induces a conformational change in the protein that propagates to the catalytic site, triggering conversion of GTP into cGMP. This review focuses on recent research that takes this basic information forward to the beginnings of a quantitative depiction of NO signal transduction, analogous to that achieved for other major transmitters. At its foundation is an explicit enzyme-linked receptor mechanism for NO-activated guanylyl cyclase that replicates all its main properties. In cells, NO signal transduction is subject to additional, activity-dependent modifications, notably through receptor desensitization and changes in the activity of cGMP-hydrolyzing phosphodiesterases. The measurement of these parameters under varying conditions in rat platelets has made it possible to formulate a cellular model of NO-cGMP signaling. The model helps explain cellular responses to NO and their modification by therapeutic agents acting on the guanylyl cyclase or phosphodiesterase limbs of the pathway.

Cytochrome P450 oxidoreductase participates in nitric oxide consumption by rat brain.

In low nanomolar concentrations, NO (nitric oxide) functions as a transmitter in brain and other tissues, whereas near-micromolar NO concentrations are associated with toxicity and cell death. Control of the NO concentration, therefore, is critical for proper brain function, but, although its synthesis pathway is well-characterized, the major route of breakdown of NO in brain is unclear. Previous observations indicate that brain cells actively consume NO at a high rate. The mechanism of this consumption was pursued in the present study. NO consumption by a preparation of central glial cells was abolished by cell lysis and recovered by addition of NADPH. NADPH-dependent consumption of NO localized to cell membranes and was inhibited by proteinase K, indicating the involvement of a membrane-bound protein. Purification of this activity yielded CYPOR (cytochrome P450 oxidoreductase). Antibodies against CYPOR inhibited NO consumption by brain membranes and the amount of CYPOR in several cell types correlated with their rate of NO consumption. NO was also consumed by purified CYPOR but this activity was found to depend on the presence of the vitamin E analogue Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), included in the buffer as a precaution against inadvertent NO consumption by lipid peroxidation. In contrast, NO consumption by brain membranes was independent of Trolox. Hence, it appears that, during the purification process, CYPOR becomes separated from a partner needed for NO consumption. Cytochrome P450 inhibitors inhibited NO consumption by brain membranes, making these proteins likely candidates.

What is the real physiological NO concentration in vivo?

Clarity about the nitric oxide (NO) concentrations existing physiologically is essential for developing a quantitative understanding of NO signalling, for performing experiments with NO that emulate reality, and for knowing whether or not NO concentrations become abnormal in disease states. A decade ago, a value of about 1 microM seemed reasonable based on early electrode measurements and a provisional estimate of the potency of NO for its guanylyl cyclase-coupled receptors, which mediate physiological NO signal transduction. Since then, numerous efforts to measure NO concentrations directly using electrodes in cells and tissues have yielded an irreconcilably large spread of values. In compensation, data from several alternative approaches have now converged to provide a more coherent picture. These approaches include the quantitative analysis of NO-activated guanylyl cyclase, computer modelling based on the type, activity and amount of NO synthase enzyme contained in cells, the use of novel biosensors to monitor NO release from single endothelial cells and neurones, and the use of guanylyl cyclase as an endogenous NO biosensor in tissue subjected to a variety of challenges. All these independent lines of evidence suggest the physiological NO concentration range to be 100 pM (or below) up to approximately 5 nM, orders of magnitude lower than was once thought.

NO as a multimodal transmitter in the brain: discovery and current status.

NO operates throughout the brain as an intercellular messenger, initiating its varied physiological effects by activating specialized GC-coupled receptors, resulting in the formation of cGMP. In line with the widespread expression of this pathway, NO participates in numerous different brain functions. This review gives an account of the discovery of NO as a signalling molecule in the brain, experiments that originated in the search for a mysterious cGMP-stimulating factor released from central neurones when their NMDA receptors were stimulated, and summarizes the subsequent key steps that helped establish its status as a central transmitter. Currently, various modes of operation are viewed to underlie its diverse behaviour, ranging from very local signalling between synaptic partners (in the orthograde or retrograde directions) to a volume-type transmission whereby NO synthesized by multiple synchronous sources summate spatially and temporally to influence intermingled neuronal or non-neuronal cells, irrespective of anatomical connectivity. LINKED ARTICLES: This article is part of a themed section on Nitric Oxide 20 Years from the 1998 Nobel Prize. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.2/issuetoc.

N10 -carbonyl-substituted phenothiazines inhibiting lipid peroxidation and associated nitric oxide consumption powerfully protect brain tissue against oxidative stress.

During some investigations into the mechanism of nitric oxide consumption by brain preparations, several potent inhibitors of this process were identified. Subsequent tests revealed the compounds act by inhibiting lipid peroxidation, a trigger for a form of regulated cell death known as ferroptosis. A quantitative structure-activity study together with XED (eXtended Electron Distributions) field analysis allowed a qualitative understanding of the structure-activity relationships. A representative compound N-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)-10H-phenothiazine-10-carboxamide (DT-PTZ-C) was able to inhibit completely oxidative damage brought about by two different procedures in organotypic hippocampal slice cultures, displaying a 30- to 100-fold higher potency than the standard vitamin E analogue, Trolox or edaravone. The compounds are novel, small, drug-like molecules of potential therapeutic use in neurodegenerative disorders and other conditions associated with oxidative stress.

Concepts of neural nitric oxide-mediated transmission.

As a chemical transmitter in the mammalian central nervous system, nitric oxide (NO) is still thought a bit of an oddity, yet this role extends back to the beginnings of the evolution of the nervous system, predating many of the more familiar neurotransmitters. During the 20 years since it became known, evidence has accumulated for NO subserving an increasing number of functions in the mammalian central nervous system, as anticipated from the wide distribution of its synthetic and signal transduction machinery within it. This review attempts to probe beneath those functions and consider the cellular and molecular mechanisms through which NO evokes short- and long-term modifications in neural performance. With any transmitter, understanding its receptors is vital for decoding the language of communication. The receptor proteins specialised to detect NO are coupled to cGMP formation and provide an astonishing degree of amplification of even brief, low amplitude NO signals. Emphasis is given to the diverse ways in which NO receptor activation initiates changes in neuronal excitability and synaptic strength by acting at pre- and/or postsynaptic locations. Signalling to non-neuronal cells and an unexpected line of communication between endothelial cells and brain cells are also covered. Viewed from a mechanistic perspective, NO conforms to many of the rules governing more conventional neurotransmission, particularly of the metabotropic type, but stands out as being more economical and versatile, attributes that presumably account for its spectacular evolutionary success.

Nitric oxide as a multimodal brain transmitter.

One of the simplest molecules in existence, nitric oxide, burst into all areas of biology some 30 years ago when it was established as a major signalling molecule in the cardiovascular, nervous and immune systems. Most regions of the mammalian brain synthesise nitric oxide and it has many diverse roles both during development and in adulthood. Frequently, nitric oxide synthesis is coupled to the activation of NMDA receptors and its physiological effects are mediated by enzyme-linked receptors that generate cGMP. Generally, nitric oxide appears to operate in two main modes: first, in a near synapse-specific manner acting either retrogradely or anterogradely and, second, when multiple nearby sources are active simultaneously, as a volume transmitter enabling signalling to diverse targets irrespective of anatomical connectivity. The rapid diffusibility of nitric oxide and the efficient capture of fleeting, subnanomolar nitric oxide concentrations by its specialised receptors underlie these modes of operation.

Tonic and phasic nitric oxide signals in hippocampal long-term potentiation.

Nitric oxide (NO) participates in long-term potentiation (LTP) and other forms of synaptic plasticity in many different brain areas but where it comes from and how it acts remain controversial. Using rat and mouse hippocampal slices, we tested the hypothesis that tonic and phasic NO signals are needed and that they derive from different NO synthase isoforms. NMDA increased NO production in a manner that was potently inhibited by three different neuronal NO synthase (nNOS) inhibitors. Tonic NO could be monitored after sensitizing guanylyl cyclase-coupled NO receptors, allowing the very low ambient NO concentrations to be detected by cGMP measurement. The levels were unaffected by inhibition of NMDA receptors, nNOS, or the inducible NO synthase (iNOS). iNOS was also undetectable in protein or activity assays. Tonic NO was susceptible to agents inhibiting endothelial NO synthase (eNOS) and was missing in eNOS knock-out mice. The eNOS knock-outs exhibited a deficiency in LTP resembling that seen in wild-types treated with a NO synthase inhibitor. LTP in the knock-outs could be fully restored by supplying a low level of NO exogenously. Inhibition of nNOS also caused a major loss of LTP, particularly of late-LTP. Again, exogenous NO could compensate, but higher concentrations were needed compared with those restoring LTP in the eNOS knock-outs. It is concluded that tonic and phasic NO signals are both required for hippocampal LTP and the two are generated, respectively, by eNOS and nNOS, the former in blood vessels and the latter in neurons.