Abstracts

Adenosine is an inhibitory neuromodulator which recently has been shown to decrease seizure activity in a kindling model of epilepsy. In the hippocampus adenosine A[sub]1 [/sub]receptor activation results in decreased neurotransmitter release and hyperpolarization of neurons. Although the mechanisms that control the release of adenosine are unknown, a decrease in intracellular pH is common to many adenosine-releasing stimuli. Increases in brain pCO[sub]2 [/sub]decrease intracellular pH and may result in adenosine release. Here we have investigated the effects of increased CO[sub]2[/sub] on adenosine release, neurotransmission, and intracellular pH changes in area CA1 of rat hippocampal slices.
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. Extracellular adenosine levels were measured using a double-barreled subtractive adenosine sensor (Dale, J Physiol., 1998). The adenosine sensor was placed across the s. pyramidale in CA1. Intracellular pH measurements were made using BCECF-AM loaded slices imaged on a Zeiss 510 2-photon microscope.
When hippocampal slices were exposed to aCSF acidified with 10% CO[sub]2[/sub] and 26 mM bicarbonate there was a significant increase in the extracellular concentration of adenosine. This increase in extracellular adenosine concentration was accompanied by a decrease in fEPSP amplitude and a decrease in BCECF fluorescence. The depression of fEPSP amplitude was attenuated by application of theophylline, an adenosine receptor antagonist. When hippocampal slices were exposed to 10% CO[sub]2[/sub] and 52mM bicarbonate (pH similar to control) extracellular adenosine levels appeared to decrease. Application of 10 % CO[sub]2[/sub] and 52mM bicarbonate did not alter fEPSP or change BCECF fluorescence.
Based on these studies it can be concluded that exposure of hippocampal slices to increased CO[sub]2[/sub] levels, as would occur during seizure or hypoxia, causes an increase in the release of adenosine into the extracellular milieu of the slice. Increased adenosine release due to elevated CO[sub]2[/sub] is mediated by the acidification which accompanies increased CO[sub]2[/sub] levels. Adenosine levels decrease upon addition of 10% CO[sub]2[/sub] and 52mM bicarbonate. This may be due to additional buffering capacity which can reduce physiological pH transients and thus reduce adenosine release, or to inhibition of high-voltage-activated calcium currents by bicarbonate. This study also suggests that the sedative and anticonvulsive effects of hypercapnia, and the proconvulsive effects of hypocapnia, may be due to changes in extracellular adenosine levels.
[Supported by: NIH grant 29173.]