The dynamics of single spike-evoked adenosine release in the cerebellum

The purine adenosine is a potent neuromodulator in the brain, with roles in a number of diverse physiological and pathological processes. Modulators such as adenosine are difficult to study as once released they have a diffuse action (which can affect many neurones) and, unlike classical neurotransmitters, have no inotropic receptors. Thus rapid postsynaptic currents (PSCs) mediated by adenosine (equivalent to mPSCs) are not available for study. As a result the mechanisms and properties of adenosine release still remain relatively unclear. We have studied adenosine release evoked by stimulating the parallel fibres in the cerebellum. Using adenosine biosensors combined with deconvolution analysis and mathematical modelling, we have characterised the release dynamics and diffusion of adenosine in unprecedented detail. By partially blocking K+ channels, we were able to release adenosine in response to a single stimulus rather than a train of stimuli. This allowed reliable sub-second release of reproducible quantities of adenosine with stereotypic concentration waveforms that agreed well with predictions of a mathematical model of purine diffusion. We found no evidence for ATP release and thus suggest that adenosine is directly released in response to parallel fibre firing and does not arise from extracellular ATP metabolism. Adenosine release events showed novel short-term dynamics, including facilitated release with paired stimuli at millisecond stimulation intervals but depletion-recovery dynamics with paired stimuli delivered over minute time scales. These results demonstrate rich dynamics for adenosine release that are placed, for the first time, on a quantitative footing and show strong similarity with vesicular exocytosis

Density, heterogeneity and deformability of red cells as markers of clinical severity in hereditary spherocytosis

Altres ajuts: This work was generated within the European Reference Network on Rare Hematological Diseases (ERN-EuroBloodNet) - FPA No. 739541Hereditary spherocytosis (HS) originates from defective anchoring of the cytoskeletal network to the transmembrane protein complexes of the red blood cell (RBC). Red cells in HS are characterized by membrane instability and reduced deformability and there is marked heterogeneity in disease severity among patients. To unravel this variability in disease severity, we analyzed blood samples from 21 HS patients with defects in ankyrin, band 3, á-spectrin or β-spectrin using red cell indices, eosin-5- maleimide binding, microscopy, the osmotic fragility test, Percoll density gradients, vesiculation and ektacytometry to assess cell membrane stability, cellular density and deformability. Reticulocyte counts, CD71 abundance, band 4.1 a:b ratio, and glycated hemoglobin were used as markers of RBC turnover. We observed that patients with moderate/severe spherocytosis have short-living erythrocytes of low density and abnormally high intercellular heterogeneity. These cells show a prominent decrease in membrane stability and deformability and, as a consequence, are quickly removed from the circulation by the spleen. In contrast, in mild spherocytosis less pronounced reduction in deformability results in prolonged RBC lifespan and, hence, cells are subject to progressive loss of membrane. RBC from patients with mild spherocytosis thus become denser before they are taken up by the spleen. Based on our findings, we conclude that RBC membrane loss, cellular heterogeneity and density are strong markers of clinical severity in spherocytosis

Sleep-wake sensitive mechanisms of adenosine release in the basal forebrain of rodents : an in vitro study

Adenosine acting in the basal forebrain is a key mediator of sleep homeostasis. Extracellular adenosine concentrations increase during wakefulness, especially during prolonged wakefulness and lead to increased sleep pressure and subsequent rebound sleep. The release of endogenous adenosine during the sleep-wake cycle has mainly been studied in vivo with microdialysis techniques. The biochemical changes that accompany sleep-wake status may be preserved in vitro. We have therefore used adenosine-sensitive biosensors in slices of the basal forebrain (BFB) to study both depolarization-evoked adenosine release and the steady state adenosine tone in rats, mice and hamsters. Adenosine release was evoked by high K+, AMPA, NMDA and mGlu receptor agonists, but not by other transmitters associated with wakefulness such as orexin, histamine or neurotensin. Evoked and basal adenosine release in the BFB in vitro exhibited three key features: the magnitude of each varied systematically with the diurnal time at which the animal was sacrificed; sleep deprivation prior to sacrifice greatly increased both evoked adenosine release and the basal tone; and the enhancement of evoked adenosine release and basal tone resulting from sleep deprivation was reversed by the inducible nitric oxide synthase (iNOS) inhibitor, 1400 W. These data indicate that characteristics of adenosine release recorded in the BFB in vitro reflect those that have been linked in vivo to the homeostatic control of sleep. Our results provide methodologically independent support for a key role for induction of iNOS as a trigger for enhanced adenosine release following sleep deprivation and suggest that this induction may constitute a biochemical memory of this state

On the Public-Private School Achievement Debate

On July 14, 2006, the U. S. Department of Education’s National Center for Education Statistics (NCES) released a study that compared the performance in reading and math of 4th and 8th-graders attending private and public schools. Using information from a nationwide, representative sample of public and private school students collected in 2003 as part of the ongoing National Assessment of Educational Progress (NAEP), the NCES study reported that the performance of students attending private schools was superior to that of students attending public schools. But after statistical adjustments were made for student characteristics, the private school advantage among 4th-graders was reported to give way to a 4.5-point public school advantage in math and school-sector parity in reading. After the same adjustments were made for 8th-graders, private schools retained a 7-point advantage in reading but achieved only parity in math. In this paper, we argue that NCES’s measures of student characteristics were flawed by inconsistent classification across the public and private sectors and by the inclusion of factors open to school influence. Utilizing the same data as the original study but substituting better measures of student characteristics, improved, alternative models identify a private school advantage in 11 out of 12 public-private comparisons. In 8th-grade math, the private school advantage varies between 3 and 6.5 test points; in reading, it varies between 9 and 12.5 points. Among 4th graders, in math, parity is observed in one model, but private schools outperform public schools by 2 and 3 points in the other two models; in 4th-grade reading, private schools have an advantage that ranges from 7 to 10 points. However, although the alternative models constitute an improvement on the NCES model, no conclusions should be drawn as to causal relationships from these or any other results based on NAEP test scores, because they are too fragile to be used for such purposes. Inferring causality from observations at one point in time is highly problematic.

A three-enzyme microelectrode sensor for detecting purine release from central nervous system

As the purities, in particular adenosine, are important signaling agents in the nervous system we have devised a new biosensor for directly measuring their production in real time during physiological activity. Our amperometric adenosine biosensor is made by entrapping 3 enzymes (xanthine oxidase, purine nucleoside phosphorylase and adenosine deaminase) in a composite lactobionamide and amphiphillic polypyrrole matrix around a Pt microelectrode. The resulting sensors are small (25-100 pm diameter), fast responding (10-90% rise time, 2+/-0.23 s), sensitive (100-222 mA M(-1.)cm(-2)) and stable (100% activity after 5 days). The sensor was used in vivo to demonstrate the spatial localization of release of adenosine from Xenopus embryo spinal cord during fictive swimming. (C) 2002 Elsevier Science B.V. All rights reserved.</p

Adenosine release in nucleus tractus solitarii does not appear to mediate hypoxia-induced respiratory depression in rats

The time course of adenosine release in the nucleus tractus solitarii (NTS) and ventrolateral medulla (VLM) during acute systemic hypoxia was investigated in the anaesthetised rat by means of amperometric enzymatic sensors. It was found that acute hypoxia induced a significant delayed increase in adenosine level (reaching levels as high as 5 μM) in the NTS and that hypoxia-induced release of adenosine was similar at various regions of the NTS along its rostro-caudal axis. Significantly smaller or no increases in adenosine levels at all in response to hypoxia were observed in the VLM. The increase in adenosine level in the NTS occurred during reoxygenation after the termination of the hypoxic challenge and was accompanied by a smaller increase in inosine concentration. At the dorsal surface of the brainstem, only release of inosine was detected following acute hypoxia. Addition of the ecto-5′-nucleotidase inhibitor α,β-methylene ADP (200 μM) to the dorsal surface of the brainstem completely abolished the signal evoked by hypoxia, suggesting that the inosine arose from adenosine that was produced in the extracellular space by the prior release of ATP. This study indicates that following systemic hypoxia, adenosine levels in the NTS increase to a significantly greater extent than in the VLM. However, the increase in adenosine concentration in the NTS occurs too late to be responsible for the hypoxia-induced depression of the respiratory activity

Modulation of the release of endogenous adenosine by cannabinoids in the myenteric preparation of the guinea-pig plexus-longitudinal muscle ileum

Original article can be found at: http://www.nature.com/bjp/index.html Copyright British Pharmacological Society and Nature Publishing Group. DOI: 10.1038/sj.bjp.0704985 [Full text of this article is not available in the UHRA]Peer reviewe