Star spangled manner: astrocytes and neurons contribute to adenosine release in the hippocampus

Neurons in the brain signal to each other by the release of chemical neurotransmitters. These are concentrated in vesicles at the presynaptic terminal and following a calcium influx the vesicles release the transmitter to act at postsynaptic receptors. In this respect adenosine is unusual, as it can be released from presynaptic vesicles, but an increase in extracellular adenosine may also result from other mechanisms. Adenosine produced in a cell can efflux passively by membrane transport proteins called equilibrative nucleoside transporters (ENTs) and extracellular adenosine can be produced from the hydrolysis of ATP by extracellularly localised enzymes called ectonucleotidases. To further complicate matters, ATP itself can be released from cells by vesicular release or through membrane channels formed of connexin. Understanding the mechanisms and function of adenosine in the brain is, therefore of great interest due to its widespread presence and diverse roles as a modulatory neurotransmitter. A major action of adenosine is the inhibition of excitatory transmission via its action at presynaptic A1 receptors. Indeed, in pathological conditions such as stroke and epilepsy, an increase in adenosine release acts to reduce neuronal activity, resulting in a neuro-protective effect. (Boison, 2008). Another physiological role of adenosine seems to be as a somnogen, whereby an increase in extracellular levels of this neurotransmitter during wakefulness leads to the promotion of sleep associated neuronal network oscillations (Brown et al. 2012). Different adenosine release mechanisms have been known and described for some time (Latini & Pedata, 2001), and it is also well known that release characteristics vary by preparation, brain area and stimulus; release may occur in response to glutamatergic agonists, ischaemia or electrical stimulation. While the situation appears complicated, past efforts to discriminate the contribution of different components were also made more difficult by the limitations of contemporaneous techniques; these included indirectly inferring adenosine levels by determining the degree of induced presynaptic inhibition, or by the use of microdialysis or radiolabelling assays. Recently, however, the development of new techniques such as transgenic mouse models, multiphoton imaging and detection methods, together with an increased acceptance of a significant role for non-neuronal cells in brain function, has made it a potentially exciting and fruitful time to be studying adenosine in the brain. Using a transgenic dnSNARE mouse mutant where astrocytic vesicular release is selectively impaired, Pascual et al. (2005) demonstrated that astrocytic vesicular ATP release was a source of adenosine that induced hippocampal heterosynaptic depression. Another study, using calcium imaging and selective neuronal patch clamp methods, concluded a predominant role for adenosine release via ENTs underlying synaptic depression (Lovatt et al. 2012). In this issue of The Journal of PhysiologyWall & Dale (2013) used combined electrophysiological recordings with adenosine- sensitive microelectrode biosensors in a hippocampal slice preparation. These biosensors have a tip diameter of 50 μm or less and can be inserted into the slice preparation to electrochemically detect adenosine, and permit real-time measurements with a high temporal resolution. They found that adenosine was released following Schaffer collateral stimulation and that this release was stimulation frequency-dependent with a peak at the relatively low frequency of 5 Hz. Extracellular adenosine concentration increase was dependent on ionotropic glutamate receptor activation and was also calcium dependent. The adenosine increase was inhibited by pharmacological inhibition of ectonucleotidase, indicating a contribution from ATP hydrolysis. However, in hippocampal slices from a transgenic mouse model lacking ectonucleotidase, adenosine release could still be measured following synaptic stimulation. This release was inhibited by pharmacological block of ENTs, indicating two parallel release mechanisms. To investigate whether adenosine also came from different cellular sources, the authors used the astrocytic metabolic poison fluoroacetate. Inhibiting the contribution of astrocytes resulted in a change in kinetics in the measured adenosine increases following synaptic stimulation so that rise and decay were more rapid. To further define the astrocytic mechanism, experiments were then conducted in dnSNARE mice where vesicular release, including that of ATP, is inhibited. Confirming the effect seen with fluoroacetate, the kinetics of adenosine release were more rapid compared to wild-type mice, indicating the loss of a slow astrocytic vesicular release component. Figure 1 Glutamate released by Schaffer collateral stimulation activates pyramidal neurones to release adenosine via equilibrative nucleotide transporters (ENT), and astrocytes to release ATP, which is hydrolysed by ectonucleotidase (ENase) to adenosine. Combining pharmacological, electrophysiological and microelectrode biosensor techniques therefore shows that, in the hippocampal slice preparation, stimulation of the Shaffer collateral induces an increase in adenosine, which results from a contribution from both neurones and astrocytes, which in turn display different temporal characteristics. This is, therefore, another example of astrocyte–neuron interaction in the brain, where both cell types act to increase the levels of the same transmitter, with the astrocytic component ostensibly extending the time period in which adenosine levels are elevated. The study provides answers to many questions about hippocampal adenosine release, yet, despite the small diameter of the biosensors, these results cannot tell us about the spatial cellular and subcellular specificity of the release. The difference in kinetics of neuronal and astrocytic adenosine hints at different physiological targets and roles; for example, one source might preferentially target synaptic or extrasynaptic receptors, or primarily target either neuronal or astrocytic networks. Addressing such questions may therefore require the further refinement and development of new techniques to detect and monitor the effects of adenosine.