To re-examine how the basal extracellular concentration of adenosine is regulated in acutely isolated cerebellar slices we have combined electrophysiological and microelectrode biosensor measurements. In almost all cases, synaptic transmission was tonically inhibited by adenosine acting via A 1 receptors. By contrast, in most slices, the biosensors did not measure an adenosine tone but did record a spatially non-uniform extracellular tone of the downstream metabolites (inosine and hypoxanthine). Most of the extracellular hypoxanthine arose from the metabolism of inosine by ecto-purine nucleoside phosphorylase (PNP). Adenosine kinase was the major determinant of adenosine levels, as its inhibition increased both adenosine concentration and A 1 receptor-mediated synaptic inhibition. Breakdown of adenosine by adenosine deaminase was the major source of the inosine/hypoxanthine tone. However adenosine deaminase played a minor role in determining the level of adenosine at synapses, suggesting a distal location. Blockade of adenosine transport (by NBTI/dipyridamole) had inconsistent effects on basal levels of adenosine and synaptic transmission. Unexpectedly, application of NBTI/dipyridamole prevented the efflux of adenosine resulting from block of adenosine kinase at only a subset of synapses. We conclude that there is spatial variation in the functional expression of NBTI/dipyridamole-sensitive transporters. The increased spatial and temporal resolution of the purine biosensor measurements has revealed the complexity of the control of adenosine and purine tone in the cerebellum.

The purine adenosine is an extracellular signalling molecule involved in a large number of physiological and pathological conditions throughout the mammalian brain. However little is known about how adenosine release and its subsequent clearance change during brain development. We have combined electrophysiology and microelectrode biosensor measurements to investigate the properties of adenosine signalling at early stages of cerebellar development, when parallel fibre-Purkinje cell synapses have recently been formed (postnatal days 9-12). At this stage of development, we could detect little or no inhibitory A 1 receptor tone in basal conditions and during trains of stimuli. Addition of pharmacological agents, to inhibit adenosine clearance, had only minor effects on synaptic transmission suggesting that under basal conditions, the concentration of adenosine moving in and out of the extracellular space is small. Active adenosine release was stimulated with hypoxia and trains of electrical stimuli. Although hypoxia released significant concentrations of adenosine, the release was delayed and slow. No adenosine release could be detected following electrical stimulation in the molecular layer. In conclusion, at this stage of development, although adenosine receptors and the mechanisms of adenosine clearance are present there is very little adenosine release.

Publisher statement: "NOTICE: this is the author's version of a work that was accepted for publication in Neuropharmacology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Neuropharmacology, VOL 61, ISSUE 4, 2011, DOI: 10.1016/j.neuropharm.2011 AbstractThe purine adenosine plays an important role in a number of physiological and pathological processes and is neuroprotective during hypoxia and ischemia. The major effect of adenosine is to suppress network activity via the activation of A 1 receptors. Here we report that in immature cerebellar slices, the activation of A 1 receptors has variable effects on parallel fibre synaptic transmission, ranging from zero depression to an almost complete abolition of transmission. Concentrationresponse curves suggest that the heterogeneity of inhibition stems from differences in A 1 receptor properties which could include coupling to downstream effectors. There is less variation in the effects of adenosine at parallel fibre synapses in slices from older rats and thus adenosine signalling appears developmentally regulated.In the cerebellum, hypoxia increases the concentration of extracellular adenosine leading to the activation of A 1 receptors (at adenosine-sensitive parallel fibre synapses) and the suppression of glutamate release. It would be predicted that the synapses that were insensitive to adenosine would be less depressed by hypoxia and thus maintain function during metabolic stress. However those synapses which were insensitive to adenosine were rapidly inhibited by hypoxia via a mechanism which was not reversed by blocking A 1 receptors. Thus another mechanism must be responsible for the hypoxia-mediated depression at these synapses. These different mechanisms of depression maybe important for cell survival and for maintenance of cerebellar function following oxygen starvation.3