Metabolic regulation of endogenous adenosine release from single neurons.

The mechanisms that regulate adenosine release in the brain are not well understood. The present study investigated the hypothesis that individual neurons can generate and release sufficient adenosine to regulate their synaptic inputs. We utilized the whole-cell recording technique to apply enzyme inhibitors and nucleotides directly into the cytoplasm of single rat hippocampal CA1 pyramidal neurons. Cytoplasmic delivery of adenosine induced the release of sufficient adenosine to inhibit excitatory synaptic inputs. However, intracellular delivery of nucleotides and enzyme inhibitors failed to increase adenosine receptor-mediated inhibition. These data suggest that while pyramidal neurons in the hippocampus are capable of releasing large amounts of adenosine into the extracellular space, they do not readily form adenosine from endogenous sources.

The role of cyclic AMP as a precursor of extracellular adenosine in the rat hippocampus.

Extracellular adenosine 3':5'-cyclic monophosphate (cAMP) is a potential source of the inhibitory neuromodulator adenosine in the brain. Previous work has demonstrated that cAMP, which is formed intracellularly, can be transported into the extracellular space and subsequently catabolized to adenosine. However, the physiological conditions under which cAMP release might lead to adenosine formation and activation of adenosine receptors are not well understood. In this study we demonstrate that superfusion of hippocampal slices with cAMP or forskolin led to the formation of extracellular adenosine which activated adenosine receptors in a manner comparable to that seen with adenosine superfusion. In contrast, application of brief pulses of cAMP onto the cell bodies of CA1 pyramidal neurons failed to produce an adenosine receptor-mediated response, while application of brief pulses of adenosine or AMP elicited significant responses. These data suggest that large, prolonged increases in extracellular cAMP levels can result in the formation of extracellular adenosine and the activation of adenosine receptors, but brief increases in cAMP levels in the vicinity of individual neurons cannot. These findings imply that increases in cAMP levels may lead to relatively slow increases in extracellular adenosine, as opposed to the fast, spatially restricted increases that would occur following the release of other adenine nucleotides.

Modulation of excitatory synaptic transmission by adenosine released from single hippocampal pyramidal neurons.

Adenosine is a potent neuromodulator in the CNS, but the mechanisms that regulate adenosine concentrations in the extracellular space remain unclear. The present study demonstrates that increasing the intracellular concentration of adenosine in a single hippocampal CA1 pyramidal neuron selectively inhibits the excitatory postsynaptic potentials in that cell. Loading neurons with high concentrations of adenosine via the whole-cell patch-clamp technique did not affect the GABAA-mediated inhibitory postsynaptic potentials, the membrane resistance, or the holding current, whereas it significantly increased the adenosine receptor-mediated depression of excitatory postsynaptic currents. The effects of adenosine could not be mimicked by an agonist at the intracellular adenosine P-site, but the effects could be antagonized by a charged adenosine receptor antagonist and by adenosine deaminase, demonstrating that the effect was mediated via adenosine acting at extracellular adenosine receptors. The effect of adenosine loading was not blocked by BaCl2 and therefore was not caused by an adenosine-activated postsynaptic potassium conductance. Adenosine loading increased the paired-pulse facilitation ratio, demonstrating that the effect was mediated by presynaptic adenosine receptors. Finally, simultaneous extracellular field recordings demonstrated that the increase in extracellular adenosine was confined to excitatory synaptic inputs to the loaded cell. These data demonstrate that elevating the intracellular concentration of adenosine in a single CA1 pyramidal neuron induces the release of adenosine into the extracellular space in such a way that it selectively inhibits the excitatory inputs to that cell, and the data support the general conclusion that adenosine is a retrograde messenger used by pyramidal neurons to regulate their excitatory input.

Purinoceptors in the Central Nervous System.

New exciting developments on the occurrence and functional role of purinoceptors in mammalian brain were presented at the session "Purinoceptors in the central nervous system" chaired by Flaminio Cattabeni and Tom Dunwiddie at the Purines '96 international conference. The focus of the session were topics of recent interest, including the sources and mechanisms involved in ATP and adenosine release during physiological neurotransmission in hippocampus, the brain expression of the recently cloned P2 receptors, and the role of the various adenosine receptor subtypes in brain protection from neurodegeneration associated with trauma-, ischemia-and excessive excitatory amino acid neurotransmission. New important insights into the mechanisms responsible for the formation and release of adenosine into the extracellular space were provided by data obtained by Dunwiddie and coworkers in hippocampal pyramidal neurons. These data may have functional implications for the role of purines in modulation of synaptic plasticity and long-term potentiation in this brain area, and hence in cognitive functions. Buell provided an updated overview on the cloning, molecular characteristics and brain expression of various ligand-gated P2X purinoceptors; although the functional role of these receptors in mammalian brain still awaits elucidation, their widespread distribution in the nervous system strongly suggests that ATP-mediated events are more prevalent and important in brain than expected. Pedata presented data on the functional interrelationships between adenosine and glutamate in the brain, and also provided evidence for alterations of the reciprocal regulation between these two systems in aged brain, which may have important implications for both ischemia-and trauma-associated neurodegenerative events and senescence-associated cognitive impairment. Finally, von Lubitz provided novel data on the molecular mechanisms likely to be at the basis of the brain protective effects associated with the chronic stimulation of the adenosine A3 receptor, further confirming that this receptor represents a crucial target for the development of new antiischemic and antineurodegenerative therapeutic agents.

The role of acetate as a potential mediator of the effects of ethanol in the brain.

Acetate is the primary product of ethanol catabolism and can accumulate in the blood at concentrations of up to 2 mM following ethanol consumption. It has been suggested that some of the pharmacological actions of ethanol are mediated via acetate, which can lead indirectly to the release of endogenous adenosine. In the present experiments this hypothesis was tested by examining the effects of exogenous sodium acetate on the physiology of hippocampal slices from rat brain. Acetate had no significant effect on intracellular responses recorded from CA1 pyramidal neurons or on extracellular field potentials evoked from the either the CA1 region or the dentate gyrus. There was also no significant difference in responses to the adenosine receptor antagonist theophylline in CA1 pyramidal neurons recorded using intracellular filling solutions containing potassium acetate, KCl, or potassium methylsulfate. These results suggest that the presence of acetate, either in the extracellular medium or within an intracellular electrode, does not induce a significant increase in adenosine receptor activation in the hippocampus.