Functional Applications of Stable Tau Oligomers in Cell Biology and Electrophysiology Studies.

Aggregated tau protein plays a key role in the pathogenesis of neurodegenerative tauopathies including Alzheimer's disease. Soluble, low-molecular-weight tau oligomers are formed early in disease processes and are thought to have toxic functions that disrupt neuronal function. The dynamic and transient nature of tau oligomers complicates in vitro functional studies to explore the mechanistic links between oligomer formation and neurodegeneration. We have previously described a method of producing stable and structurally characterized oligomers that maintain their oligomeric conformation and prevent further aggregation. This method allows for the flexibility of stabilizing tau oligomers by specifically labelling cysteine residues with fluorescent or colorless maleimide conjugates. Here, we describe the functional applications of these preformed stable tau oligomers in cell biology and electrophysiological studies. These investigations allow real-time insights into the cellular uptake of exogenous tau oligomers and their functional effects in the recipient cells.

Adenosine is released during thalamic oscillations to provide negative feedback control.

Physiological oscillations in the cortico-thalamo-cortical loop occur during processes such as sleep, but these can become dysfunctional in pathological conditions such as absence epilepsy. The purine neuromodulator adenosine can act as an endogenous anticonvulsant: it is released into the extracellular space during convulsive seizures to activate A1 receptors suppressing on-going activity and delaying the occurrence of the next seizure. However, the role of adenosine in thalamic physiological and epileptiform oscillations is less clear. Here we have combined immunohistochemistry, electrophysiology, and fixed potential amperometry (FPA) biosensor measurements to characterise the release and actions of adenosine in thalamic oscillations measured in rodent slices. In the thalamus, A1 receptors are highly expressed particularly in the ventral basal (VB) thalamus and reticular thalamic nucleus (nRT) supporting a role for adenosine signalling in controlling oscillations. In agreement with previous studies, both adenosine and adenosine A1 receptor agonists inhibited thalamic oscillations in control (spindle-like) and in epileptic conditions. Here we have shown for the first time that both control and epileptiform oscillations are enhanced (i.e., increased number of oscillatory cycles) by blocking A1 receptors consistent with adenosine release occurring during oscillations. Although increases in extracellular adenosine could not be directly detected during control oscillations, clear increases in adenosine concentration could be detected with a biosensor during epileptiform oscillation activity. Thus, adenosine is released during thalamic oscillations and acts via A1 receptors to feedback and reduce thalamic oscillatory activity.

Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression.

The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, benzyloxy-cyclopentyladenosine (BnOCPA), is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPA's unique and exquisitely selective activation of Gob among the six Gαi/o subtypes, and in the absence of ß-arrestin recruitment. BnOCPA thus demonstrates a highly-specific Gα-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective Gα agonism.

The comparative effects of mGlu5 receptor positive allosteric modulators VU0409551 and VU0360172 on cognitive deficits and signalling in the sub-chronic PCP rat model for schizophrenia.

In schizophrenia, mGlu5 receptor hypofunction has been linked with neuropathology and cognitive deficits, making it an attractive therapeutic target. The cognitive impairment associated with schizophrenia remains an unmet clinical need, with existing antipsychotics primarily targeting positive symptoms, with weaker and more variable effects on cognitive deficits. Using the sub-chronic phencyclidine rat model, widely shown to mimic the cognitive impairment and neuropathology of schizophrenia, we have investigated two mGlu5 receptor positive allosteric modulators (PAMs), VU0409551 and VU0360172. We compared the efficacy of these compounds in restoring cognitive deficits and, since these two PAMs have reportedly distinct signalling mechanisms, changes in mGlu5 receptor signalling molecules AKT and MAPK in the PFC. Although not effective at 0.05 and 1 mg/kg, cognitive deficits were significantly alleviated by both PAMs at 10 and 20 mg/kg. The compounds appeared to have differential effects on the scPCP-induced increases in AKT and MAPK phosphorylation: VU0409551 induced a significant decrease in expression of p-AKT, whereas VU0360172 had this effect on p-MAPK levels. Thus, the beneficial effects of PAMs on scPCP-induced cognitive impairment are accompanied by at least partial reversal of scPCP-induced elevated levels of p-MAPK and p-AKT, whose dysfunction is strongly implicated in schizophrenia pathology. These promising data imply an important role for mGlu5 receptor signalling pathways in improving cognition in the scPCP model and provide support for mGlu5 receptor PAMs as a possible therapeutic intervention for schizophrenia.

Examining Local Cell-to-Cell Signalling in the Kidney Using ATP Biosensing.

Cell-to-cell communication is an essential process for the efficient function of cells and tissues. Central to this is the purinergic transmission of purines, with ligands such as adenosine triphosphate (ATP). Altered cell-to-cell communication, and in particular changes in the paracrine release of extracellular ATP, plays crucial roles in pathophysiological conditions, such as diabetes. ATP biosensing provides a reliable, real-time measurement of local extracellular ATP concentrations. This allows the detection of altered ATP release, which underlies the progression of inflammation and fibrosis and is a potential therapeutic target. Here we describe in a step-by-step basis how to utilize sensitive microelectrode biosensors to detect low, real-time concentrations of ATP, in vitro.

Acute ethanol exposure has bidirectional actions on the endogenous neuromodulator adenosine in rat hippocampus.

BACKGROUND AND PURPOSE: Ethanol is a widely used recreational drug with complex effects on physiological and pathological brain function. In epileptic patients, the use of ethanol can modify seizure initiation and subsequent seizure activity with reports of ethanol being both pro- and anticonvulsant. One proposed target of ethanol's actions is the neuromodulator adenosine, which is released during epileptic seizures to feedback and inhibit the occurrence of subsequent seizures. Here, we investigated the actions of acute ethanol exposure on adenosine signalling in rat hippocampus. EXPERIMENTAL APPROACH: We have combined electrophysiology with direct measurements of extracellular adenosine using microelectrode biosensors in rat hippocampal slices. KEY RESULTS: We found that ethanol has bidirectional actions on adenosine signalling: depressant concentrations of ethanol (50 mM) increased the basal extracellular concentration of adenosine under baseline conditions, leading to the inhibition of synaptic transmission, but it inhibited adenosine release during evoked seizure activity in brain slices. The reduction in activity-dependent adenosine release was in part produced by effects on NMDA receptors, although other mechanisms also appeared to be involved. Low concentrations of ethanol (10-15 mM) enhanced pathological network activity by selectively blocking activity-dependent adenosine release. CONCLUSIONS AND IMPLICATIONS: The complex dose-dependent actions of ethanol on adenosine signalling could in part explain the mixture of pro-convulsant and anticonvulsant actions of ethanol that have previously been reported.

Reducing Extracellular Ca2+ Induces Adenosine Release via Equilibrative Nucleoside Transporters to Provide Negative Feedback Control of Activity in the Hippocampus.

Neural circuit activity increases the release of the purine neuromodulator adenosine into the extracellular space leading to A1 receptor activation and negative feedback via membrane hyperpolarization and inhibition of transmitter release. Adenosine can be released by a number of different mechanisms that include Ca2+ dependent processes such as the exocytosis of ATP. During sustained pathological network activity, ischemia and hypoxia the extracellular concentration of calcium ions (Ca2+) markedly falls, inhibiting exocytosis and potentially reducing adenosine release. However it has been observed that reducing extracellular Ca2+ can induce paradoxical neural activity and can also increase adenosine release. Here we have investigated adenosine signaling and release mechanisms that occur when extracellular Ca2+ is removed. Using electrophysiology and microelectrode biosensor measurements we have found that adenosine is directly released into the extracellular space by the removal of extracellular Ca2+ and controls the induced neural activity via A1 receptor-mediated membrane potential hyperpolarization. Following Ca2+ removal, adenosine is released via equilibrative nucleoside transporters (ENTs), which when blocked leads to hyper-excitation. We propose that sustained action potential firing following Ca2+ removal leads to hydrolysis of ATP and a build-up of intracellular adenosine which then effluxes into the extracellular space via ENTs.

Modeling microelectrode biosensors: free-flow calibration can substantially underestimate tissue concentrations.

Microelectrode amperometric biosensors are widely used to measure concentrations of analytes in solution and tissue including acetylcholine, adenosine, glucose, and glutamate. A great deal of experimental and modeling effort has been directed at quantifying the response of the biosensors themselves; however, the influence that the macroscopic tissue environment has on biosensor response has not been subjected to the same level of scrutiny. Here we identify an important issue in the way microelectrode biosensors are calibrated that is likely to have led to underestimations of analyte tissue concentrations. Concentration in tissue is typically determined by comparing the biosensor signal to that measured in free-flow calibration conditions. In a free-flow environment the concentration of the analyte at the outer surface of the biosensor can be considered constant. However, in tissue the analyte reaches the biosensor surface by diffusion through the extracellular space. Because the enzymes in the biosensor break down the analyte, a density gradient is set up resulting in a significantly lower concentration of analyte near the biosensor surface. This effect is compounded by the diminished volume fraction (porosity) and reduction in the diffusion coefficient due to obstructions (tortuosity) in tissue. We demonstrate this effect through modeling and experimentally verify our predictions in diffusive environments.NEW & NOTEWORTHY Microelectrode biosensors are typically calibrated in a free-flow environment where the concentrations at the biosensor surface are constant. However, when in tissue, the analyte reaches the biosensor via diffusion and so analyte breakdown by the biosensor results in a concentration gradient and consequently a lower concentration around the biosensor. This effect means that naive free-flow calibration will underestimate tissue concentration. We develop mathematical models to better quantify the discrepancy between the calibration and tissue environment and experimentally verify our key predictions.

Activity-Regulated Cytoskeleton-Associated Protein Controls AMPAR Endocytosis through a Direct Interaction with Clathrin-Adaptor Protein 2.

The activity-regulated cytoskeleton-associated (Arc) protein controls synaptic strength by facilitating AMPA receptor (AMPAR) endocytosis. Here we demonstrate that Arc targets AMPAR to be internalized through a direct interaction with the clathrin-adaptor protein 2 (AP-2). We show that Arc overexpression in dissociated hippocampal neurons obtained from C57BL/6 mouse reduces the density of AMPAR GluA1 subunits at the cell surface and reduces the amplitude and rectification of AMPAR-mediated miniature-EPSCs (mEPSCs). Mutations of Arc, that prevent the AP-2 interaction reduce Arc-mediated endocytosis of GluA1 and abolish the reduction in AMPAR-mediated mEPSC amplitude and rectification. Depletion of the AP-2 subunit µ2 blocks the Arc-mediated reduction in mEPSC amplitude, an effect that is restored by reintroducing µ2. The Arc-AP-2 interaction plays an important role in homeostatic synaptic scaling as the Arc-dependent decrease in mEPSC amplitude, induced by a chronic increase in neuronal activity, is inhibited by AP-2 depletion. These data provide a mechanism to explain how activity-dependent expression of Arc decisively controls the fate of AMPAR at the cell surface and modulates synaptic strength, via the direct interaction with the endocytic clathrin adaptor AP-2.

Combined electrophysiological and biosensor approaches to study purinergic regulation of epileptiform activity in cortical tissue.

BACKGROUND: Cortical brain slices offer a readily accessible experimental model of a region of the brain commonly affected by epilepsy. The diversity of recording techniques, seizure-promoting protocols and mutant mouse models provides a rich diversity of avenues of investigation, which is facilitated by the regular arrangement of distinct neuronal populations and afferent fibre pathways, particularly in the hippocampus. NEW METHOD AND RESULTS: We have been interested in the regulation of seizure activity in hippocampal and neocortical slices by the purines, adenosine and ATP. Via the use of microelectrode biosensors we have been able to measure the release of these important neuroactive compounds simultaneously with on-going epileptiform activity, even of brief durations. In addition, detailed numerical analysis and computational modelling has produced new insights into the kinetics and spatial distribution of elevations in purine concentration that occur during seizure activity. COMPARISON AND CONCLUSIONS: Such an approach allows the spatio-temporal characteristics of neurotransmitter/neuromodulator release to be directly correlated with electrophysiological measures of synaptic and seizure activity, and can provide greater insight into the role of purines in epilepsy.

Localized adenosine signaling provides fine-tuned negative feedback over a wide dynamic range of neocortical network activities.

Although the patterns of activity produced by neocortical networks are now better understood, how these states are activated, sustained, and terminated still remains unclear. Negative feedback by the endogenous neuromodulator adenosine may potentially play an important role, as it can be released by activity and there is dense A1 receptor expression in the neocortex. Using electrophysiology, biosensors, and modeling, we have investigated the properties of adenosine signaling during physiological and pathological network activity in rat neocortical slices. Both low- and high-rate network activities were reduced by A1 receptor activation and enhanced by block of A1 receptors, consistent with activity-dependent adenosine release. Since the A1 receptors were neither saturated nor completely unoccupied during either low- or high-rate activity, adenosine signaling provides a negative-feedback mechanism with a wide dynamic range. Modeling and biosensor experiments show that during high-rate activity increases in extracellular adenosine concentration are highly localized and are uncorrelated over short distances that are certainly<500 µm. Modeling also predicts that the slow rise of the purine waveform cannot be from diffusion from distal release sites but more likely results from uptake and metabolism. The inability to directly measure adenosine release during low-rate activity, although it is present, is probably a consequence of small localized increases in adenosine concentration that are rapidly diminished by diffusion and active removal mechanisms. Saturation of such removal mechanisms when higher concentrations of adenosine are released results in the accumulation of inosine, explaining the strong purine signal during high-rate activity.

Visfatin reduces gap junction mediated cell-to-cell communication in proximal tubule-derived epithelial cells.

BACKGROUND/AIMS: In the current study we examined if the adipocytokine, visfatin, alters connexin-mediated intercellular communication in proximal tubule-derived epithelial cells. METHODS: The effects of visfatin (10-200ng/mL) on cell viability and cytotoxicity in HK2-cells were assessed by MTT, crystal violet and lactate dehydrogenase assays. Western blot analysis was used to confirm expression of Cx26, Cx40 and Cx43. The effect of visfatin (10-200ng/mL) on TGF-ß1 secretion was confirmed by ELISA, and the effects of both TGF-ß1 (2-10ng/mL) and visfatin (10-200ng/mL) on connexin expression were assessed by western blot. Functional intercellular communication was determined using transfer of Lucifer Yellow and paired-whole cell patch clamp electrophysiology. RESULTS: In low glucose (5mM), visfatin (10-200ng/mL) did not affect membrane integrity, cytotoxicity or cell viability at 48hrs, but did evoke a concentration-dependent reduction in Cx26 and Cx43 expression. The expression of Cx40 was unaffected. At 48hrs, visfatin (10-200ng/mL) increased the secretion of TGF-ß1 and the visfatin-evoked changes in connexin expression were mimicked by exogenous application of the pro-fibrotic cytokine (2-10ng/ml). Visfatin reduced dye transfer between coupled cells and decreased functional conductance, with levels falling by 63% as compared to control. Although input resistance was increased following visfatin treatment by 166%, the change was not significant as compared to control. The effects of visfatin on Cx-expression and cell-coupling were blocked in the presence of a TGF-ß1 specific neutralizing antibody. CONCLUSIONS: The adipocytokine visfatin selectively evoked a non-toxic reduction in connexin expression in HK2-cells. The loss in gap-junction associated proteins was mirrored by a loss in functional conductance between coupled cells. Visfatin increased TGF-ß secretion and the pattern of change for connexins expression was mimicked by exogenous application of TGF-ß1. The effect of visfatin on Cx-expression and dye transfer were negated in the presence of a TGF-ß1 neutralising antibody. These data suggest that visfatin reduces connexin-mediated intercellular communication in proximal tubule-derived epithelial cells via a TGF-ß dependent pathway.

Enhancement of kinase selectivity in a potent class of arylamide FMS inhibitors.

Structure-activity relationship (SAR) studies on a highly potent series of arylamide FMS inhibitors were carried out with the aim of improving FMS kinase selectivity, particularly over KIT. Potent compound 17r (FMS IC50 0.7 nM, FMS cell IC50 6.1 nM) was discovered that had good PK properties and a greater than fivefold improvement in selectivity for FMS over KIT kinase in a cellular assay relative to the previously reported clinical candidate 4. This improved selectivity was manifested in vivo by no observed decrease in circulating reticulocytes, a measure of bone safety, at the highest studied dose. Compound 17r was highly active in a mouse pharmacodynamic model and demonstrated disease-modifying effects in a dose-dependent manner in a strep cell wall-induced arthritis model of rheumatoid arthritis in rats.

Neuronal transporter and astrocytic ATP exocytosis underlie activity-dependent adenosine release in the hippocampus.

The neuromodulator adenosine plays an important role in many physiological and pathological processes within the mammalian CNS. However, the precise mechanisms of how the concentration of extracellular adenosine increases following neural activity remain contentious. Here we have used microelectrode biosensors to directly measure adenosine release induced by focal stimulation in stratum radiatum of area CA1 in mouse hippocampal slices. Adenosine release was both action potential and Ca²âº dependent and could be evoked with low stimulation frequencies and small numbers of stimuli. Adenosine release required the activation of ionotropic glutamate receptors and could be evoked by local application of glutamate receptor agonists. Approximately 40% of stimulated-adenosine release occurred by translocation of adenosine via equilibrative nucleoside transporters (ENTs). This component of release persisted in the presence of the gliotoxin fluoroacetate and thus results from the direct release of adenosine from neurons. A reduction of adenosine release in the presence of NTPDase blockers, in slices from CD73(-/-) and dn-SNARE mice, provides evidence that a component of adenosine release arises from the extracellular metabolism of ATP released from astrocytes. This component of release appeared to have slower kinetics than the direct ENT-mediated release of adenosine. These data suggest that activity-dependent adenosine release is surprisingly complex and, in the hippocampus, arises from at least two distinct mechanisms with different cellular sources.

Adenosine A1 receptor activation mediates the developmental shift at layer 5 pyramidal cell synapses and is a determinant of mature synaptic strength.

During the first postnatal month glutamatergic synapses between layer 5 pyramidal cells in the rodent neocortex switch from an immature state exhibiting a high probability of neurotransmitter release, large unitary amplitude and synaptic depression to a mature state with decreased probability of release, smaller unitary amplitude and synaptic facilitation. Using paired recordings, we demonstrate that the developmental shift in release probability at synapses between rat somatosensory layer 5 thick-tufted pyramidal cells is mediated by a higher and more heterogeneous activation of presynaptic adenosine A1 receptors. Immature synapses under control conditions exhibited distributions of coefficient of variation, failure rate and release probability that were almost coincident with the A1 receptor blocked condition; however, mature synapses under control conditions exhibited much broader distributions that spanned those of both the A1 receptor agonized and antagonized conditions. Immature and mature synapses expressed A1 receptors with no observable difference in functional efficacy and therefore the heterogeneous A1 receptor activation seen in the mature neocortex appears due to increased adenosine concentrations that vary between synapses. Given the central role demonstrated for A1 receptor activation in determining synaptic amplitude and the statistics of transmission between mature layer 5 pyramidal cells, the emplacement of adenosine sources and sinks near the synaptic terminal could constitute a novel form of long-term synaptic plasticity.

Deletion of ecto-5'-nucleotidase (CD73) reveals direct action potential-dependent adenosine release.

Purinergic signaling is a highly complex system of extracellular communication involved in many physiological and pathological functions in the mammalian brain. Its complexity stems from the multitude of purine receptor subtypes and endogenous purine receptor ligands (including ATP, ADP, UTP, UDP, and adenosine). Potentially all of these ligands could be directly released, and some could also arise from extracellular metabolism. A widely held consensus is that, except under pathological conditions, extracellular adenosine arises only from ectoATPase-mediated metabolism of previously released ATP. Here, we have used mice that lack the CD73 gene (encoding ecto-5'-nucleotidase that converts AMP to adenosine) to test whether action potential-dependent adenosine release in the cerebellum depends on prior ATP release. Surprisingly, we have uncovered two parallel pathways of adenosine release: one that is indirect via glutamate receptor-dependent release of ATP and a second of equal amplitude that has no dependence on prior release of ATP and thus represents the direct release of adenosine. This component of adenosine release is blocked by bafilomycin and modulated by mGlu4 receptor activation, strongly supporting adenosine release by exocytosis from parallel fibers. Our findings are a major step in understanding the mechanisms of adenosine release and are likely to have implications for all aspects of physiology where adenosine plays a key modulatory role.

Receptor-mediated modulation of activity-dependent adenosine release in rat cerebellum.

Although the neuromodulator adenosine plays an important role in many central nervous system physiological and pathological processes, the properties and mechanisms of extracellular adenosine production are still unclear. In previous work, we determined that two forms of adenosine release can be evoked in the molecular layer of the cerebellum: one independent of ionotropic glutamate receptor activation (evoked by a train of stimuli) and one mainly dependent on the activation of ionotropic glutamate receptors (evoked by a single stimulus in 4-aminopyridine). Here we have investigated how these different forms of adenosine release are modulated by metabotropic receptors (A(1), GABA(B) and mGlu4). Although both types of adenosine release are inhibited by the activation of metabotropic receptors, single stimulus-evoked release was much more potently inhibited suggesting differential coupling between receptors and adenosine release mechanisms. Metabotropic receptor antagonists revealed that endogenous A(1) receptor activation plays the major role in controlling adenosine release and determine the relationship between stimulus strength and adenosine release. The major mechanism of modulation is through control of ionotropic glutamate receptor activation with block of metabotropic receptors inducing glutamate receptor-dependent adenosine release. In contrast to metabotropic receptor agonists, which inhibit adenylyl cyclase, activation of adenylyl cyclase (with forskolin) increased both glutamate receptor-dependent and independent adenosine release. This is the first time that the control of adenosine release by endogenous modulators has been studied and like classical neurotransmitters, adenosine release is controlled by an interplay of presynaptic modulators. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

The sodium-potassium pump controls the intrinsic firing of the cerebellar Purkinje neuron.

In vitro, cerebellar Purkinje cells can intrinsically fire action potentials in a repeating trimodal or bimodal pattern. The trimodal pattern consists of tonic spiking, bursting, and quiescence. The bimodal pattern consists of tonic spiking and quiescence. It is unclear how these firing patterns are generated and what determines which firing pattern is selected. We have constructed a realistic biophysical Purkinje cell model that can replicate these patterns. In this model, Na(+)/K(+) pump activity sets the Purkinje cell's operating mode. From rat cerebellar slices we present Purkinje whole cell recordings in the presence of ouabain, which irreversibly blocks the Na(+)/K(+) pump. The model can replicate these recordings. We propose that Na(+)/K(+) pump activity controls the intrinsic firing mode of cerbellar Purkinje cells.

Optimization of a potent class of arylamide colony-stimulating factor-1 receptor inhibitors leading to anti-inflammatory clinical candidate 4-cyano-N-[2-(1-cyclohexen-1-yl)-4-[1-[(dimethylamino)acetyl]-4-piperidinyl]phenyl]-1H-imidazole-2-carboxamide (JNJ-28312141).

A class of potent inhibitors of colony-stimulating factor-1 receptor (CSF-1R or FMS), as exemplified by 8 and 21, was optimized to improve pharmacokinetic and pharmacodynamic properties and potential toxicological liabilities. Early stage absorption, distribution, metabolism, and excretion assays were employed to ensure the incorporation of druglike properties resulting in the selection of several compounds with good activity in a pharmacodynamic screening assay in mice. Further investigation, utilizing the type II collagen-induced arthritis model in mice, culminated in the selection of anti-inflammatory development candidate JNJ-28312141 (23, FMS IC(50) = 0.69 nM, cell assay IC(50) = 2.6 nM). Compound 23 also demonstrated efficacy in rat adjuvant and streptococcal cell wall-induced models of arthritis and has entered phase I clinical trials.

A population of immature cerebellar parallel fibre synapses are insensitive to adenosine but are inhibited by hypoxia.

The 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. Concentration-response 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 may be important for cell survival and for maintenance of cerebellar function following oxygen starvation.