Release of ATP from hippocampal slices during spreading depression
Date
1999
Authors
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ORCID
Type
Degree Level
Masters
Abstract
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.
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Citation
Degree
Master of Science (M.Sc.)
Department
Physiology
Program
Physiology