Intracellular acidification causes adenosine release during states of hyperexcitability in the hippocampus

Abstract

Decreased pH increases extracellular adenosine in CNS regions as diverse as hippocampus and ventral medulla. However, thus far there is no clear consensus whether the critical pH change is a decrease in intracellular and/or extracellular pH. Previously we showed that a decrease in extracellular pH is necessary and a decrease in intracellular pH alone is not sufficient, to increase extracellular adenosine in an acute hippocampal slice preparation. Here we explored further the role of intracellular pH under different synaptic conditions in the hippocampal slice. When synaptic excitability was increased, either during gamma-aminobutyric acid type A receptor blockade in CA1 or after the induction of persistent bursting in CA3, a decrease in intracellular pH alone was now sufficient to: 1) elevate extracellular adenosine concentration, 2) activate adenosine A1 receptors, 3) decrease excitatory synaptic transmission (CA1), and 4) attenuate burst frequency in an in vitro seizure model (CA3). Hippocampal slices obtained from adenosine A1 receptor knockout mice did not exhibit these pH-mediated effects on synaptic transmission, further confirming the role of adenosine acting at the adenosine A1 receptor. Taken together, these data strengthen and add significantly to the evidence outlining a change in pH as an important stimulus influencing extracellular adenosine. In addition, we identify conditions under which intracellular pH plays a dominant role in regulating extracellular adenosine concentrations.

FIG. 1.

Intracellular acidification releases adenosine and inhibits synaptic transmission. A: field excitatory postsynaptic potentials (fEPSPs) in CA1 stratum radiatum. Left: averaged fEPSP traces during control (black) and 20 mM propionic acid exposure (gray). Center: averaged fEPSP traces during 100 μM picrotoxin (black) and picrotoxin + propionic acid exposure (gray). Right: averaged fEPSP traces during picrotoxin + 250 μM theophylline (black) and propionic acid exposure (gray). Scale bars = 50 ms and 0.5 mV. B: time course of propionic acid alone (black circles, n = 12), propionic acid in picrotoxin pretreatment (gray triangles, n = 23), propionic acid in picrotoxin, and theophylline pretreatment (gray squares, n = 12). C: average change in fEPSP slope (***P < 0.001). D: change in adenosine levels when propionic acid was added under control conditions (n = 5) and when propionic acid was added after γ-aminobutyric acid type A (GABA_A) receptors were blocked with picrotoxin (n = 7, **P < 0.01, Student's t-test).

FIG. 2.

Changes in BCECF [2′,7′-bis(2-carboxyethyl)-5,6-carboxyfluorescein] fluorescence in CA1 pyramidal cells. Percentage change in BCECF fluorescence was measured during exposure to propionic acid (n = 6), picrotoxin (n = 7), picrotoxin + propionic acid (n = 8), and high buffering power buffer + picrotoxin + propionic acid (n = 6). *P < 0.05 compared with the change in BCECF fluorescence during picrotoxin + propionic acid exposure( ANOVA, Fisher's protected least-significant difference test).

FIG. 3.

Reducing intracellular acidification attenuates the effects of propionic acid during GABA_A receptor blockade. A: fEPSPs in CA1 stratum radiatum. Averaged fEPSP traces during 100 μM picrotoxin (black) and picrotoxin + propionic acid exposure in high buffering power buffer (gray). Scale bars = 50 ms and 0.5 mV. B: time course of propionic acid effects. Propionic acid in picrotoxin pretreatment (black circles, n = 23; also shown in Fig. 1), propionic acid in picrotoxin, and high buffering power buffer pretreatment (open circles, n = 7). C: average change in fEPSP slope was appreciably attenuated in high bicarbonate buffer (***P < 0.001, compared with inhibition of fEPSP slope caused by 20 mM propionic acid in the presence of picrotoxin alone; propionate and picrotoxin data also shown in Fig. 1C). D: propionic acid treatment caused adenosine release when GABA_A receptors were blocked with picrotoxin (n = 7), but did not if a high buffering power buffer was used (n = 3, **P < 0.01, Student's t-test).

FIG. 4.

Adenosine A₁ receptor (A₁R) knockout genotype eliminates the effects of intracellular acidification on fEPSP slope during GABA_A receptor blockade. fEPSPs from CA1 stratum radiatum in hippocampal slices obtained from wild-type mice (A) and A₁R knockout mice (B) during control conditions (black) and during exposure to 100 μM adenosine (gray). fEPSPs from wild-type mice (C) and A₁R knockout mice (D) during exposure to picrotoxin (black) and propionic acid and picrotoxin (gray). Scale bars = 50 ms and 0.5 mV. E: time course of propionic acid effect on fEPSP in wild-type mice (light-gray), wild-type mice in the presence of theophylline (dark gray), and A₁R knockout mice (black). F: in wild-type mice propionic acid decreased fEPSP slope when GABA_A receptors were blocked with picrotoxin (n = 10). In A₁R knockout mice this effect was abolished (n = 10, *P < 0.05, Student's t-test).

FIG. 5.

Propionic acid attenuates epileptiform activity in area CA3 via A₁Rs. Extracellular recordings of synchronized bursting in area CA3. A: propionic acid exposure reversibly attenuates bursting. Inset, left, 1: example of a single burst recorded during control conditions (scale bars 1 mV, 50 ms for all). 2: example of extracellular recording during propionic acid exposure, with no bursts present. (Note: exact locations of samples indicated below.) Inset, right: burst frequency during control and propionic acid exposure. B: when A₁Rs were blocked with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), burst frequency was increased compared with A, and subsequent application of propionic acid slowed ongoing bursting activity. Unlike A, where propionic acid caused a complete cessation of bursting in 5/6 slices, bursting activity slowed significantly but never ceased in the presence of DPCPX. Inset, left, 1: example of a single burst recorded during control conditions with application of DPCPX. 2: example of a single burst recorded during propionic acid with application of DPCPX, with bursts still present. Inset, right: burst frequency during control/DPCPX and propionic acid/DPCPX conditions. C: during synchronized network activity in area CA3 propionic acid causes a significant release of adenosine (n = 4, **P < 0.01), whereas under control recording conditions in area CA1 propionic acid does not cause a change in adenosine release (n = 5, P = not significant). D: propionic acid reduced burst frequency in all slices. Propionic acid alone abolished bursting in 5 of 6 slices (left column, open symbol, n = 6). In the presence of DPCPX, bursting frequency decreased but was never abolished in any slice (right column, solid symbol, n = 6).