Action potential and Ca2+ dependent adenosine release in the cerebellum: release mechanisms and signalling properties

Adenosine is a vital signalling molecule that plays important roles in normal and aberrant brain function. An abiding area of uncertainty is the cellular sources and mechanisms of adenosine release. We are now in a uniquely advantageous position to study this problem as we have developed novel technology to measure directly activity-dependent adenosine release from in-vitro rodent brain slices. Our measurements of adenosine have cast doubt on the entrenched consensus- that adenosine cannot be released by exocytosis (a universal mechanism of release). We therefore aim to demonstrate the cellular source of adenosine and whether or not direct exocytotic release of adenosine occurs in a brain region called the cerebellum. This demonstration has the potential to revolutionize our understanding of adenosine signalling by demonstrating that a hitherto largely discounted class of mechanism does indeed play a role. We shall then characterize important properties of adenosine signalling such as: how it depends upon patterns of neural activity; how it can be modulated; how adenosine diffuses to inhibit signalling between neighbouring neurones; and how far and for how long this inhibitory action extends. We shall create a detailed computational model that will aid our comprehension of how adenosine controls information flow in cerebellum.

Although adenosine is an important neuromodulator, with many physiological and pathophysiological roles, many facets of adenosine signalling remain uncertain. For example: What is the cellular source of adenosine? How is adenosine released? What are the temporal and spatial properties of adenosine signalling? To answer these questions we have combined recently developed biosensor technology and electrophysiology and identified an action potential and Ca2+ dependent form of adenosine release in the cerebellum. This mode of adenosine signalling is likely to be of considerable physiological importance as it is evoked by neural activity, it modulates transmitter release, appears to occur in multiple brain regions and can be modulated by endogenous neurotransmitters. We propose to fully define this form of adenosine release and place it within a physiological context. Thus our two primary objectives are:
1) To fully define the mechanism of cerebellar adenosine release (determine whether adenosine is released directly or as a precursor, confirm that parallel fibre activity is required for adenosine release and then determine whether adenosine is directly released from parallel fibres by exocytosis). This hypothesis led research will either confirm the current consensus (that adenosine is only released as ATP/cAMP or by reverse transport) or, as our preliminary evidence strongly suggests, overturn the consensus and provide direct evidence of adenosine exocytosis.

2) Fully characterise the signalling properties of activity-dependent adenosine release including kinetics, spatial/temporal properties and modulation by endogenous transmitters. Further insight into this potentially complex signalling system will be provided by the construction of experimentally testable computational and mathematical models of adenosine signalling in the cerebellum. This combined experimental and theoretical approach will establish, for the first time, a quantitative framework for this important signalling mechanism in the central nervous system.