ATP -a mediator of central chemoreception in brain stem

Without a mechanical aid, it is impossible to stop breathing by will power. Why? Because the levels of CO2 in inspired air and in blood are an extremely powerful drive to breathe ?if these levels go up (as they would during self-willed asphyxiation or re-breathing from a plastic bag) we breathe more powerfully and rapidly. Specialized areas of the brain stem have long been known to detect CO2 in brain and relay this information to the neural networks that control breathing. Despite this, the detailed mechanisms of CO2 detection (chemoreception), and indeed the chemosensory cells themselves remain unknown. We have recently made a breakthrough in discovering that ATP is released from these specialized areas of the brain stem during elevated CO2 and is an essential link in the chemosensory reflex. We shall now exploit this finding by using experimentally simpler in vitro preparations of the brain stem. We shall look at the mechanisms of ATP release, as these will lead us to a fundamental understanding of the mechanisms of chemoreception. Thus we shall produce detailed maps of the locations of ATP release. We shall look at the characteristics of ATP release, which may highlight the types of cells that release ATP during elevated CO2. We shall identify the ion channels within the chemosensory cells responsible for detecting changes in CO2 and ultimately causing the release of ATP. Finally we shall examine release from of ATP from isolated cells in response to CO2 to allow us to identify them.

In mammals, PCO2 in arterial blood is a powerful stimulus for respiration. Central chemoreceptors within the medulla oblongata detect brain PCO2 and if this rises enhance ventilation to ensure appropriate homeostatic control. We have shown in vivo and in vitro for the first time that ATP is released from these chemosensitive areas and constitutes an important first and causal link in the chemosensory reflex. We now propose to use in vitro preparations of the ventral medulla to study the role of ATP in central chemoreception further. Using microdisk biosensors selective for ATP, we shall precisely map the anatomical locations of ATP release during hypercapnia both in vitro (from isolated horizontal slices of the ventral medulla and en bloc neonatal brain stem preparations) and in vivo in the anaesthetized, artificially ventilated rat. This will allow us to relate the locations of ATP release to known groups of neurons in the ventral medulla. We shall examine whether ATP release depends on external Ca2+ and is sensitive to TTX as this will help to identify the types of release mechanism involved. As changes in PCO2 are very likely detected as changes in pH, we shall test the dependence of ATP release on pH-sensitive ion channels such as the TASKs, KIRs and ASICs by using a variety of ion channel blockers. The release of ATP is closely linked to the CO2 stimulus implying that ectonucleotidases must be involved in the termination of ATP-signalling. We shall therefore study the localization of the ectonucleotidases relative to ATP release sites (in situ hybridization with specific cDNA antisense probes and immunocytochemistry) and, by using specific blockers of ectoATPases, obtain direct experimental evidence both in vitro and in vivo for their involvement in the control of ATP accumulation and the physiological response to hypercapnia. Finally we shall exploit our precise knowledge of the location of the ATP-releasing chemosensitive cells to acutely isolate them from the ventral medulla. This will allow us to study CO2-evoked ATP release from single cells with microbiosensors and, by post-hoc staining with selective markers (e.g. against GFAP, neuron specific enolase, NK1 receptors, somatostatin), gain definitive evidence as to their cellular identity.