Intracellular ATP and the Purinergic Modulation of Seizure Activity
All living systems require energy for biological and molecular processes. This energy can be available in the form of the cellular energy-carrying purine molecule adenosine triphosphate (ATP). Purines such as ATP, adenosine and cAMP are essential constituents of all living cells and are important molecules for both intracellular and extracellular signalling.
The purine ribonucleoside adenosine can be formed by the catabolism of ATP and it can also be directly released from neurons, glia and endothelium. Adenosine regulates many physiological processes, especially in excitable tissues such as the heart and brain (Dale and Frenguelli, 2009; Dunwiddie, 1999; Dunwiddie and Masino, 2001; Fredholm et al., 2011; Fredholm et al., 2001). Research has shown that a decrease in energy supplies and ATP lead to increased adenosine, which in turn provide negative feedback inhibition to reduce metabolic demand to save energy (Newby et al., 1985). Many actions of adenosine either reduce the activity of excitable tissues or increase the delivery of metabolic substrates thereby helping to couple the rate of energy expenditure to the energy demand. Adenosine plays a variety of different roles as an intracellular messenger, in the brain where the expression of adenosine receptors are found in high concentrations adenosine has been implicated in both normal and pathophysiological processes, including sleep, arousal, neuroprotection and epilepsy (Stone et al., 2009).
Epilepsy is a debilitating neurological disease that affects 1 in 200 people in the UK (Bell and Sander, 2001). Typically, epileptic seizures arise as a result of abnormal, excessive electrical discharges in neurones (Cavazo and Sanchez, 2004), this causes a sudden imbalance between excitatory and inhibitory processes in the neural circuitry (Engelborghs et al., 2000), in part due to the release of inhibitory and excitatory neurotransmitters. Brain injury or stress leads to an acute surge of micromolar levels of adenosine that is beyond normal levels. This acute surge in adenosine is a consequence of increased ATP degradation and decreased adenosine clearance by acute down-regulation of adenosine kinase. These high levels of adenosine can then trigger several downstream effects such as astogliosis and seizures that all contribute to trigger subsequent epileptogenesis (Aronica et al., 2011; Boison, 2010, 2013; Boison et al., 2010; Li et al., 2008). In vitro experiments from our lad conducted in hippocampal slices show that reduction in the basal tone of adenosine by adenosine kinase is permissive to seizure generation, in addition adenosine also exhibits anticonvulsant effects in experimental models of epilepsy (Dunwiddie, 1999). Taken together, we believe it is important to further elucidate the role adenosine plays in animal models of epilepsy, importantly during seizure activity. Considering that ATP is the primary source of intracellular adenosine, it can be postulated that any change in the ATP pool level, is likely to influence adenosine production and release. Therefore, my research will be focusing on investigating the role ATP precursors have on the restoration of intracellular ATP levels and the implications for adenosine production and release. To do this I use manipulations that regulate the availability of purine salvage pathway metabolites and possibly improve bioenergenetic and functional recovery in the brain. Slices are pre-incubated in a combination of D-ribose, the sugar backbone of ATP, and adenine the free purine base an important component of nucleic acids. Additionally, slices are also pre-incubated with creatine, which acts as a buffer of intracellular ATP breakdown. These compounds will help to elucidate whether they influence adenosine release and seizure activity in electrophysiological recordings in the CA1 region of the rat hippocampus. Field excitatory post synaptic (fEPSP) and spontaneoues seizure activity recordings are made from the Schaffer-collateral pathway in the hippocampus of the rat brain. The role of the adenosine receptors can be studied either pharmacologically or by altering the expression of the receptor protein. The use of agonists allows the characterisation of the type of responses that might be elicited and the use of antagonists determines the influence of the endogenous agonist and under what circumstances.
Nissl staining and tri-synaptic circuit of the hippocampus. A) The hippocampus is divided into four regions: Cornu Ammonis 1-3 (CA1- CA3) regions and the dentate gyrus (DG) indicated by the straight arrow. Also seen is the the polymorphic layer (arrowhead). B) Input into the hippocampus from the entorhinal cortex via the perforant pathway synapse on the granule cells in the DG. This message is then passed along to the CA3 pyramidal cells.Axons from the CA3 region synapse on CA1 pyramidal cells via the Schaffer collateral fibres. Finally, the axons of the CA1 pyramidal cells project to the subiculum and the entorhinal cortex (EC).
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Prof. Bruno Frenguelli
School of Life Sciences
University of Warwick