Release of ATP from hippocampal slices during spreading depression

A SD wave represents a short lasting and reversible breakdown of ion gradients in brain cells. These waves occur only in brain tissue and are, therefore, a hallmark of this tissue. SD waves radiate out of the damaged ischemic core into the penumbra and healthy tissue. In the penumbra propagation of SD waves may lead to irreversible damage of brain cells. In healthy tissue SD waves activate microglial cells, the resident macrophage cells of the brain. The signaling pathways leading to activation of microglia are not known. It is known that extracellular ATP is a powerful activator of microglial cells. If ATP is released during SD waves it might be an evidence that ATP serves as a signal for transformation of microglia from resting to activated form. Our experiments were designed to find out whether ATP is released during SD wave. SD waves were artificially elicited in the rat hippocampal slices (400 µm) by bath application of 100 µM ouabain or by micro-injection of 1.2 M KCl into stratum radiatum of the CA1 region. Ouabain triggered SD waves with an amplitude 6.5±5.2 mV and a duration 11.4±5.2 min that appeared with a 7.5±2.8 min delay after switching normal solution to the solution containing ouabain. The ATP outflow from six slices was measured in the perfusate samples collected before and after application of ouabain. The concentration of ATP in the samples was determined with a luciferase-luciferin system. The basal outflow from six slices averaged 3.3±1.6 pmol/slice/min. Ouabain caused a transient overflow of ATP 10.4±4.4 pmo/slice/min. The increase in ATP outflow started before the appearance of the SD wave. The experiments failed to determine the ATP release from a single hippocampal slice during SD wave elicited by micro-injection of high KCl. The sensitivity of luciferin-luciferase system was not sufficient for determination of the ATP concentration in the perfusate samples collected before, during and after SD wave. Manipulations involving slow-down of the rate of ATP hydrolysis by ecto enzymes, improvement of the experimental chamber, and concentration of the perfusate samples did not improve the sensitivity. In a separate set of experiments it was demonstrated that approximately 2/3 of extracellular ATP injected into the hippocampal slice were hydrolyzed by nonspecific phosphatases and ectonucleotidases. The observed increase of the ATP release during ouabain-evoked SD wave is not definitive evidence that SD waves cause the release of ATP, because of the possibility that ouabain by itself rather than SD might cause the release of the ATP