Key roles of the histaminergic system in sleep-wake regulation

Histamine plays an important role in mediating wakefulness in mammals. Based on the findings from gene-manipulated mice, we provide several lines of evidence showing the roles of the histaminergic system in the somnogenic effects of prostaglandin (PG) D2 and adenosine, and in the arousal effects of PGE2 and orexin. PGD2 activates DP1 receptors (R) to promote sleep by stimulating them to release adenosine. The released adenosine activates adenosine A2AR and subsequently excites the ventrolateral preoptic area (VLPO), one of the sleep centers in the anterior hypothalamus. VLPO neurons then send inhibitory signals to downregulate the histaminergic tuberomammillary nucleus (TMN), which contributes to arousal. A1R is expressed in histaminergic neurons of the rat TMN. Adenosine in the TMN inhibits the histaminergic system via A1R and promotes non–rapid eye movement sleep. Conversely, both endogenous PGE2 and orexin activate the histaminergic system through EP4R and OX-2R, respectively, to promote wakefulness via histamine H1R. Furthermore, the arousal effect of ciproxifan, H3R antagonist, depends on the activation of histaminergic systems. These findings indicate that VLPO and TMN regulate sleep and wakefulness by means of a “flip-flop” mechanism operating in an anti-coincident manner during sleep–wake state transitions.

     

Modulating P1 Adenosine Receptors in Disease Progression of SOD1G93A Mutant Mice

Amyotrophic lateral sclerosis (ALS) is a fatal progressing neurodegenerative disease; to date, despite the intense research effort, only two therapeutic options, with very limited effects, are available. The purinergic system has been indicated as a possible new therapeutic target for ALS, but the results are often contradictory and generally confused. The present study was designed to determine whether P1 adenosine receptor ligands affected disease progression in a transgenic model of ALS. SOD1^G93A mice were chronically treated, from presymptomatic stage, with a selective adenosine A_2A receptor agonist (CGS21680), antagonist (KW6002) or the A₁ receptor antagonist DPCPX. Body weight, motor performance and survival time were evaluated. The results showed that neither the stimulation nor the blockade of adenosine A_2A receptors modified the progressive loss of motor skills or survival of mSOD1^G93A mice. Conversely, blockade of adenosine A₁ receptors from the presymptomatic stage significantly attenuated motor disease progression and induced a non-significant increase of median survival in ALS mice. Our data confirm that the modulation of adenosine receptors can elicit very different (and even opposite) effects during the progression of ALS course, thus strengthens the importance of further studies to elucidated their real therapeutic potential in this pathology.

     

CIRCADIAN RHYTHMS AND SLEEP IN HUMAN AGING

This issue of Chronobiology International is dedicated to the age-related changes in circadian rhythms as they occur in humans. It seems timely to give an overview of the knowledge and hypotheses on these changes now that we enter a century in which the number and percentage of elderly in the population will be unprecedented. Although we should take care not to follow the current tendency to think of old age as a disease—ignoring the fine aspects of being old—there is definitely an age-related increase in the risk of a number of conditions that are at least uncomfortable.

Circadian rhythms have been attributed adaptive values that usually go unnoticed, but can surface painfully clear when derangements occur. Alterations in the regulation of circadian rhythms are thought to contribute to the symptoms of a number of conditions for which the risk is increased in old age (e.g., sleep disturbances, dementia, and depression). A multidisciplinary approach to investigate the mechanisms of age-related changes in circadian regulation eventually may result in treatment strategies that will improve the quality of life of the growing number of elderly.

Although diverse topics are addressed in this issue, the possible mechanisms by which a deranged circadian timing system may be involved in sleep disturbances receives the most attention. This seems appropriate in view of the numerous studies that have addressed this relation in the last decade and also because of the high frequency and strong impact of sleep disturbances in the elderly. This introduction to the special issue first briefly addresses the impact of disturbed sleep in the elderly to show that the development of therapeutic methods other than the currently available pharmacological treatments should be given high priority. I believe that chronobiological insights may play an important role in the development of rational therapeutical methods.(Chronobiology International, 17(3), 233–243, 2000)     

Regulation of tumor infiltrated innate immune cells by adenosine

Cancer has the ability to escape the immune system using different molecular actors. Adenosine is known to be involved in mechanisms which control inflammatory reactions and prevent excessive immune response. This purine nucleoside can be translocated from the cell or produced in the extracellular space by 5′-ectonucleotidases. Once bound to its receptors on the surface of immune effector cells, adenosine activates various molecular pathways, which lead to functional inhibition of the cell or its death. Some tumors are infiltrated by the different cells of immune system but are able to use adenosine as an immunosuppressive molecule and thus inhibit immune anticancer response. This mechanism is well described on adaptive cells, but much less on innate cells. This review outlines major effects of adenosine on innate immune cells, its consequences on cancer progression, and possible ways to block the adenosine-dependent immunosuppressive effect.

     

The ATP-cytokine-adenosine hypothesis: How the brain translates past activity into sleep

Tumor necrosis factor alpha (TNF), interleukin-1 beta (IL1), and other cytokines are involved in non-rapid eye movement sleep (NREM) regulation under physiological and inflammatory conditions. Brain levels of IL1 and TNF increase with prolonged wakefulness. Injection of exogenous IL1 or TNF, mimicking sleep loss, induces sleepiness, excess sleep, fatigue, poor cognition, and enhanced sensitivity to pain. These symptoms characterize the syndrome associated with sleep loss. Extracellular ATP released during neuro- and glio-transmission, acting via purine P2 receptors on glia, releases IL1 and TNF. This extracellular ATP mechanism may provide an index of activity used by the brain to keep track of prior wakefulness. Prolonged wakefulness is associated with enhanced neuronal activity. TNF and IL1, in turn, act on neurons to change their intrinsic properties and sensitivities to neurotransmitters and neuromodulators such as adenosine and glutamate. Such actions change network input–output properties (i.e. state shift). State oscillations, for instance, occur within cortical columns and are responsive to TNF. Sleep is thus viewed as a local usedependent process regulated in part by cytokines. Further, state oscillations are viewed as a fundamental process of any neuronal/glia network. To investigate these hypotheses we developed an in vitro neuronal/glia culture system exhibiting field potential oscillations and have mathematically modeled the local use-dependent view of sleep initiation. These views have profound implications for sleep pathologies and function.

     

Neurotransmitter receptor availability in the rat brain is constant in a 24 hour-period

Wakefulness and sleep are fundamental characteristics of the brain. We, therefore, hypothesized that transmitter systems contribute to their regulation and will exhibit circadian alterations.

We assessed the concentration of various neurotransmitter receptors and transporters including adenosinergic (A₁AR, A_2AAR, and ENT1), dopaminergic (D₁R, D₂R, and DAT), and serotonergic (5-HT_2AR) target proteins. Adult male Sprague Dawley rats were used and maintained in a 12 h light: 12 h dark cycle (lights on from 07:00 h to 19:00 h). We measured receptor and transporter concentrations in different brain regions, including caudate putamen, basal forebrain, and cortex in 4 hour-intervals over a 24 hour-period using quantitative in vitro autoradiography.

Investigated receptors and transporters showed no fluctuations in any of the analyzed regions using one-way ANOVA. Only in the horizontal diagonal band of Broca, the difference of A₁AR concentration between light and dark phases (t-test) as well as the cosinor analysis of the 24 hour-course were significant, suggesting that this region underlies receptor fluctuations.

Our findings suggest that the availability of the investigated neurotransmitter receptors and transporters does not undergo changes in a 24 hour-period. While there are reports on changes in adenosine and dopamine receptors during sleep deprivation, we found no changes in the investigated adenosine, dopamine, and serotonin receptors during regular and undisturbed day-night cycles.     

The Role of Glial Adenosine Receptors in Neural Resilience and the Neurobiology of Mood Disorders

Adenosine receptors were classified into A₁- and A₂-receptors in the laboratory of Bernd Hamprecht more than 25 years ago. Adenosine receptors are instrumental to the neurotrophic effects of glia cells. Both microglia and astrocytes release after stimulation via adenosine receptors factors that are important for neuronal survival and growth. Neuronal resilience is now considered as of pivotal importance in the neurobiology of mood disorders and their treatment. Both sleep deprivation and electroconvulsive therapy, two effective therapeutic measures in mood disorders, are associated with an increase of adenosine and upregulation of adenosine A₁-receptors in the brain. Parameters closely related to adenosine receptor activation such as cerebral metabolic rate and delta power in the sleep EEG provide indirect evidence that adenosinergic signaling may be associated with the therapeutic response to these measures. Thus, neurotrophic effects evoked by adenosine receptors might be important in the mechanism of action of ECT and perhaps also sleep deprivation.     

Real-time measurement of adenosine and ATP release in the central nervous system

This brief review recounts how, stimulated by the work of Geoff Burnstock, I developed biosensors that allowed direct real-time measurement of ATP and adenosine during neural function. The initial impetus to create an adenosine biosensor came from trying to understand how ATP and adenosine-modulated motor pattern generation in the frog embryo spinal cord. Early biosensor measurements demonstrated slow accumulation of adenosine during motor activity. Subsequent application of these biosensors characterized real-time release of adenosine in in vitro models of brain ischaemia, and this line of work has recently led to clinical measurements of whole blood purine levels in patients undergoing carotid artery surgery or stroke. In parallel, the wish to understand the role of ATP signalling in the chemosensory regulation of breathing stimulated the development of ATP biosensors. This revealed that release of ATP from the chemosensory areas of the medulla oblongata preceded adaptive changes in breathing, triggered adaptive changes in breathing via activation of P2 receptors, and ultimately led to the discovery of connexin26 as a channel that mediates CO₂-gated release of ATP from cells.

     

Interaction between sleep mechanisms and orexin neurons

Orexin is a neuropeptide that plays a highly important role in mechanisms that regulate sleep/wake states. Lack of the orexin gene or orexin-producing neurons (orexin neurons) results in narcolepsy in several mammalian species, suggesting that orexin is an important factor for the maintenance of wakefulness. Constitutive, ectopic expression of orexin in transgenic mice resulted in severe fragmentation of non–rapid eye movement sleep, along with abnormal muscle tone regulation during REM sleep, suggesting that activity of orexin neurons should be appropriately decreased during sleep to maintain consolidated sleep states. This review will discuss the mechanisms by which the orexin system is regulated during sleep.

     

Ecstasy and sleep disturbance: Progress towards elucidating a role for the circadian system

MDMA (ecstasy) is an illicit drug which has pharmacological actions on the serotonin system, leading to a number of physiological and behavioral changes. Research conducted in both animals and humans has focused on how ecstasy use affects systems or functions in which serotonin has a regulatory role including mood, sleep and circadian rhythms. In this paper we review the evidence with respect to changes in sleep and circadian rhythms following ecstasy use. Studies of the subjective measurement of sleep have suggested that there are changes in sleep quality and duration following ecstasy use, while research utilizing objective measures including polysomnog-raphy has highlighted changes in sleep architecture following ecstasy use. Collectively these findings suggest that there are consequences associated with ecstasy use, and the implications of these findings for ecstasy users will be examined. Finally, preliminary evidence from the animal literature implicating ecstasy as having specific effects on the circadian system will be reviewed. A discussion of the limitations of the current evidence for such a claim is presented, and possible directions for future research are explored.

     

Adenosine as a sleep factor

What puts us to sleep? This question has bothered the mankind for thousands of years, but we still have no definite answer. After abandoning philosophical and religious explanations, science has adopted this question and started to examine it with experimental methods. Two early pioneers in this field, Dr. Ishimori from Japan and Drs. Pieron and Legendre from France developed the concept of hypnotoxin — a factor that accumulates during waking and puts animals and humans to sleep. They were able to show that, indeed, during deprivation of sleep, something accumulates in body — something that can be removed and will induce sleep in another individual. Later research has identified many substances that affect sleep. One of them is adenosine, which fulfils the criteria of a physiologic sleep factor.

     

The role of pannexin 1 in the purinergic regulation of synaptic transmission in mouse motor synapses

The role of pannexin 1 in the release to the extracellular space of ATP/adenosine modulating the acetylcholine (ACh) secretion was studied in mouse diaphragm motor synapses. Using neuromuscular preparations obtained from wild-type and pannexin-1 knockout mice, the miniature endplate potential (MEPPs) and evoked endplate potentials (EPPs) were recorded in combination with pharmacological modulation of P2-type ATP receptors and A₁-type adenosine receptors. Selective inhibition of A₁ receptors with DPCPX or P2 receptors with PPADS increased quantal content of EPPs in wild-type mice. MRS 2211, selective antagonist of P2Y13 receptors, produced the same effect. Activation of receptors A₁ or P2Y₁₃ by their agonists (2-CADO and IDP, respectively) decreased the EPP quantal content. It means that the activity of endogenous ATP and adenosine is synergistic and directed to depression of the ACh release. ARL67156, an inhibitor of synaptic ecto-ATPases, which blocks the hydrolysis of ATP to adenosine and increases the level of ATP in the synaptic cleft, prolonged EPPs without changing their quantal content. In pannexin-1 knockout mice there were no changes in the EPP quantal content and in other parameters of synaptic transmission as compared to wildtype mice. However, downregulation of purinergic effects with antagonists of A₁ or P2 receptors (DPCPX, PPADS, MRS 2211) did not change EPP quantal content and any other parameters of spontaneous or evoked ACh release in all cases. ARL67156 did not alter the temporal parameters of EPPs, either. Nevertheless, 2-CADO, the A₁-type receptor agonist, decreased the EPP quantal content, while the agonist of P2Y₁₃ receptors decreased the MEPP amplitude. Thus, in mice lacking pannexin 1, procedures revealing the presence and regulatory activity of synaptic ATP/adenosine did not change the parameters of synaptic transmission. The obtained data substantiate a mandatory role of pannexin 1 in the purinergic regulation of motor synapse activity by endogenous ATP/adenosine.     

Adenosine, an endogenous distress signal, modulates tissue damage and repair

Adenosine is formed inside cells or on their surface, mostly by breakdown of adenine nucleotides. The formation of adenosine increases in different conditions of stress and distress. Adenosine acts on four G-protein coupled receptors: two of them, A(1) and A(3), are primarily coupled to G(i) family G proteins; and two of them, A(2A) and A(2B), are mostly coupled to G(s) like G proteins. These receptors are antagonized by xanthines including caffeine. Via these receptors it affects many cells and organs, usually having a cytoprotective function. Joel Linden recently grouped these protective effects into four general modes of action: increased oxygen supply/demand ratio, preconditioning, anti-inflammatory effects and stimulation of angiogenesis. This review will briefly summarize what is known and what is not in this regard. It is argued that drugs targeting adenosine receptors might be useful adjuncts in many therapeutic approaches.     

腺苷和睡眠觉醒调节

腺苷作为神经调质,调节多种神经生物学功能.随觉醒时间延长,动物脑内腺苷水平逐渐增高,在睡眠期显著降低.因此,腺苷被认为是调节睡眠的内稳态因子之一.腺苷受体(receptor,R)有A1R、A2AR、A2BR和A3R四种亚型,其中A1R和A2AR与诱导睡眠相关.激活A1R可抑制促觉醒神经元诱导睡眠,也可抑制促眠神经元导致...     

Adenosine receptors as drug targets

There are four adenosine receptors, A₁, A_2A, A_2B and A₃, together forming a defined subgroup of G protein coupled receptors. They are well conserved and widely expressed. The endogenous agonist, adenosine, has a minimal concentration in body fluids (20-200 nM) that is sufficient to slightly activate the receptors where they are very highly expressed—as in the basal ganglia, on fat cells and in the kidney. Here adenosine can play a physiological role and here antagonists such as caffeine can have effects in healthy individuals. Adenosine levels rise in stress and distress (up to 30 μM in ischemia) and tend to minimize the risk for adverse outcomes by increasing energy supply and decreasing cellular work, by stimulating angiogenesis, mediating preconditioning and having multiple effects on immune competent cells. These pathophysiological roles of adenosine also offer some potential drug targets, but the fact that adenosine receptors are involved in so many processes does not simplify drug development.     

Molecular probes for the human adenosine receptors

Adenosine receptors, G protein–coupled receptors (GPCRs) that are activated by the endogenous ligand adenosine, have been considered potential therapeutic targets in several disorders. To date however, only very few adenosine receptor modulators have made it to the market. Increased understanding of these receptors is required to improve the success rate of adenosine receptor drug discovery. To improve our understanding of receptor structure and function, over the past decades, a diverse array of molecular probes has been developed and applied. These probes, including radioactive or fluorescent moieties, have proven invaluable in GPCR research in general. Specifically for adenosine receptors, the development and application of covalent or reversible probes, whether radiolabeled or fluorescent, have been instrumental in the discovery of new chemical entities, the characterization and interrogation of adenosine receptor subtypes, and the study of adenosine receptor behavior in physiological and pathophysiological conditions. This review summarizes these applications, and also serves as an invitation to walk another mile to further improve probe characteristics and develop additional tags that allow the investigation of adenosine receptors and other GPCRs in even finer detail.

     

Perinatal Caffeine,Acting on Maternal Adenosine A1 Receptors,Causes Long-Lasting Behavioral Changes in Mouse Offspring

Background

There are lingering concerns about caffeine consumption during pregnancy or the early postnatal period, partly because there may be long-lasting behavioral changes after caffeine exposure early in life.

Methodology/Principal Findings

We show that pregnant wild type (WT) mice given modest doses of caffeine (0.3 g/l in drinking water) gave birth to offspring that as adults exhibited increased locomotor activity in an open field. The offspring also responded to cocaine challenge with greater locomotor activity than mice not perinatally exposed to caffeine. We performed the same behavioral experiments on mice heterozygous for adenosine A₁ receptor gene (A₁RHz). In these mice signaling via adenosine A₁ receptors is reduced to about the same degree as after modest consumption of caffeine. A₁RHz mice had a behavioral profile similar to WT mice perinatally exposed to caffeine. Furthermore, it appeared that the mother''s genotype, not offspring''s, was critical for behavioral changes in adult offspring. Thus, if the mother partially lacked A₁ receptors the offspring displayed more hyperactivity and responded more strongly to cocaine stimulation as adults than did mice of a WT mother, regardless of their genotype. This indicates that long-term behavioral alterations in the offspring result from the maternal effect of caffeine, and not a direct effect on fetus. WT offspring from WT mother but having a A₁R Hz grandmother preserved higher locomotor response to cocaine.

Conclusions/Significance

We suggest that perinatal caffeine, by acting on adenosine A₁ receptors in the mother, causes long-lasting behavioral changes in the offspring that even manifest themselves in the second generation.     

Implication of serotonin in the control of vigilance states as revealed by knockout-mouse studies

Genetic manipulation of the 5-HT system leads to alterations of 5-HT neurotransmission and provides new opportunities to investigate the role of 5-HT in sleep regulations. Indeed, it represents an alternative to the use of pharmacological tools and, to some extent, of localized lesions of the 5-HT system, which have been, from the 1960s until recently, the main approaches to investigate this question. Homologous recombination knocking-out genes encoding various proteins involved in 5-HT neurotransmission in the mouse has recently allowed further assesment of the role of the serotonin transporter (5-HTT), the monoamine oxidase A (MAO-A), and the 5-HT1A, 5-HT1B and 5-HT2A receptors in the regulation of sleep. In 5-HT1A -/- and 5-HT1B -/- knock-out mice, Rapid Eye Movement sleep (REMs) was enhanced. Pharmacological blockade of these receptors had the same effects in wild-types. Thus, both receptor types exert a tonic inhibitory influence on REMs. In addition, 5-HT1A -/- and 5-HT1B -/- mutants were hypersensitive to 5-HT1B and 5-HT1A receptor agonists, respectively, which suggests that adaptive changes at 5-HT neurotransmission develop in knock-out animals. In the same manner, 5-HTT-/- knock-out mice exhibited increased REMs. This may be accounted for by a decrease in 5-HT1A and 5-HT1B receptor-mediated sleep regulations. In contrast, decreased REMs was observed in MAOA -/- knock-outs, a phenomenon that mimics the effect of pharmacological MAO inhibition. Finally, 5-HT2A -/- and 5-HT2C -/- mice exhibited more wakefulness and less slow wave sleep (SWS) than wild-types. These effects could not be reproduced by 5-HT2A or 5-HT2c receptor blockade in wild-types. To conclude, constitutive knock-outs undergo adaptive processes involving other proteins than those encoded by the invalidated gene, which renders interpretation of the corresponding sleep phenotype difficult. Inducible knock-outs will probably help to overcome this difficulty. Finally, combination of genetic manipulations with relevant pharmacological ones should allow further progress in the understanding of sleep mechanisms.