Abstracts

RATIONALE: Adenosine is a neuromodulator in the CNS that decreases both presynaptic release of neurotransmitter and postsynaptic excitability via the adenosine A[sub]1[/sub]receptor. While endogenous levels of adenosine inhibit synaptic transmission tonically, during times of metabolic stress (hypoxia/anoxia, ischemia, seizure, etc.) large amounts of adenosine are released. The profound inhibitory influence of this additional adenosine is thought to be neuroprotective. All of these metabolically stressful conditions, known to release adenosine, are also associated with a decrease in intracellular pH. Here we used electrophysiological recordings to test the hypothesis that intracellular pH changes cause adenosine release in the CA1 region of rat hippocampal slices.
METHODS: Hippocampal slices were prepared from 6-8 week old Sprague-Dawley rats. 400 uM slices were cut in ice cold artificial cerebrospinal fluid (aCSF) bubbled with 95% oxygen/5% carbon dioxide. Field excitatory postsynaptic potentials (fEPSPs) were evoked by stimulation of Schaffer collateral axons with a bipolar stimulating electrode and the fEPSP slope was measured with a recording electrode placed in the stratum radiatum of area CA1.
RESULTS: Under control conditions, decreasing intracellular pH with 20mM propionic acid did not appear to cause an increase in extracellular adenosine as measured by either the amplitude or slope of fEPSPs. Interestingly, however, when neuronal excitability was increased (such as during GABA[sub]A[/sub] receptor blockade with picrotoxin), a decrease in intracellular pH with propionic acid increased the release of adenosine and inhibited excitatory neurotransmission. This effect was blocked by theophylline, a non-selective adenosine antagonist, and was not present in adenosine A[sub]1[/sub] receptor knock-out mice. To try to limit the pH-altering effect of propionic acid we used a higher buffering capacity aCSF (52mM NaBicarbonate bubbled with 90% O2/ 10% CO2). This completely blocked the effect of propionic acid when applied during GABA[sub]A[/sub]providing further evidence that it is the pH change that causes the adenosine-mediated decrease in synaptic transmission. A dose response curve for 2-chloroadenosine, a non-metabolizable adenosine analogue, was performed under control conditions and in the presence of propionic acid, which showed that the pH effects could not be explained by changes in binding or receptor coupling.
CONCLUSIONS: We conclude that intracellular pH causes adenosine release in area CA1 of the hippocampus during GABA[sub]A[/sub] receptor blockade. This suggests that pH changes must occur in conjunction with other cellular events, such as increased cytosolic calcium levels, electrical activity, or reduced chloride shunting of excititory inputs, in order to release adenosine. This has implications regarding the brain[ssquote]s ability to regulate excitability during seizures and other types of heightened neuronal activity.
[Supported by: This work was supported by NIH grants R01 29173 and T32 HD41697-01.]