Modulation of excitatory synaptic transmission by adenosine released from single hippocampal pyramidal neurons

Abstract

Adenosine is a potent neuromodulator in the CNS, but the mechanisms that regulate adenosine concentrations in the extracellular space remain unclear. The present study demonstrates that increasing the intracellular concentration of adenosine in a single hippocampal CA1 pyramidal neuron selectively inhibits the excitatory postsynaptic potentials in that cell. Loading neurons with high concentrations of adenosine via the whole-cell patch-clamp technique did not affect the GABAA-mediated inhibitory postsynaptic potentials, the membrane resistance, or the holding current, whereas it significantly increased the adenosine receptor-mediated depression of excitatory postsynaptic currents. The effects of adenosine could not be mimicked by an agonist at the intracellular adenosine P-site, but the effects could be antagonized by a charged adenosine receptor antagonist and by adenosine deaminase, demonstrating that the effect was mediated via adenosine acting at extracellular adenosine receptors. The effect of adenosine loading was not blocked by BaCl2 and therefore was not caused by an adenosine-activated postsynaptic potassium conductance. Adenosine loading increased the paired-pulse facilitation ratio, demonstrating that the effect was mediated by presynaptic adenosine receptors. Finally, simultaneous extracellular field recordings demonstrated that the increase in extracellular adenosine was confined to excitatory synaptic inputs to the loaded cell. These data demonstrate that elevating the intracellular concentration of adenosine in a single CA1 pyramidal neuron induces the release of adenosine into the extracellular space in such a way that it selectively inhibits the excitatory inputs to that cell, and the data support the general conclusion that adenosine is a retrograde messenger used by pyramidal neurons to regulate their excitatory input.

Fig. 1.

The response to theophylline is proportional to the amount of adenosine in the extracellular space. A, The effect of theophylline on the excitatory postsynaptic current (EPSC) in a single neuron. Theophylline (THEO; 200 μm) was superfused during the time shown by thehorizontal bar and caused a small increase in the amplitude of the EPSC. The responses illustrated on theright were obtained during the baseline recording (a) and during the application of theophylline (b) at the time points indicated. Theophylline caused a 22% increase in the EPSC response, consistent with an 18% inhibition of the response by adenosine during the baseline recording.B, The effect of 200 μm theophylline (THEO; horizontal bar) on an adenosine-loaded cell (5 mm adenosine in the recording pipette). Theophylline caused a 395% increase in the amplitude of the EPSC, consistent with an 80% inhibition of the EPSC during the baseline. C, The effect of 200 μmtheophylline (THEO) on a cell recorded with normal filling solution in the recorded pipette (no adenosine inside) but with 30 μm exogenous adenosine (ADO) added to the bath. Exogenous adenosine inhibited the EPSC by 66% relative to the baseline, and this inhibition was reversed by theophylline. Thus, the increase in the EPSC produced by theophylline can be used to estimate the amount of adenosine-mediated inhibition of baseline responses. Each point on the graphs represents the average of five EPSCs evoked at 20 sec intervals, and each response on the right is averaged from seven consecutive sweeps.

Fig. 2.

The response to 200 μm theophylline in neurons recorded with and without 5 mm adenosine in the electrode. A, The effects of theophylline on the amplitude of the EPSC, the GABA_A receptor-mediated IPSC, membrane resistance (as determined by the current response to a fixed voltage step), and the holding current required to clamp the cell at −70 mV. Each parameter is expressed as a percentage of the pretheophylline control values, with the exception of the holding current, which is shown as the inward current response to theophylline in pA. Error bars represent SEM; **, denotes a significant difference (p < 0.01) relative to the control electrode solution. The numbers on each barindicate the number of cells recorded. B, Synaptically evoked currents from a cell recorded with a control electrode solution. The downward deflection (inward current) is the EPSC. The smaller EPSC response is the control response, and the larger response is the EPSC during application of 200 μm theophylline.C, Synaptic currents in a cell recorded with an electrode solution containing 5 mm adenosine; as inB, the larger response is the EPSC during superfusion with 200 μm theophylline. Each tracing is an average of 8–12 sweeps.

Fig. 3.

Concentration–response relationship for adenosine in the electrode filling solution. The data were transformed from the percentage of increase of the EPSC induced by theophylline to the percentage of inhibition of the EPSC by adenosine before theophylline application (assuming the response to 200 μm theophylline represents a response without any adenosine-mediated inhibition). This transformation facilitates the quantitative analysis of the data, because the maximal effect of adenosine is 100% inhibition of the EPSC response, whereas the maximal response to theophylline essentially could go to infinity. Under control conditions (no adenosine in electrode), endogenous adenosine inhibits the EPSC by ∼20%, whereas with the highest concentration of adenosine tested (15 mm), the EPSC is depressed by ∼80%. The line represents the computer-generated fit to the data according to the equation:Y = min_resp + (max_resp − min_resp)/(1 + 10^{[(logEC₅₀− X) × H]), with an EC₅₀ value of 2.12 mm and a Hill slope of 0.58. The numbers in parentheses indicate the number of cells recorded. In this and subsequent figures, *p < 0.05 and **p < 0.01 relative to 0 mm adenosine.}

Fig. 4.

Summary of the mean ± SEM inhibition of the EPSC as determined by application of adenosine antagonists under various conditions; the data were transformed, as in Figure 3.Open bars represent electrodes with 0 mmadenosine, filled bars with 5 mm adenosine, and the hatched bar is 5 mm dideoxyadenosine (DDA), a P-site agonist. The antagonists used were theophylline (Theo; 200 μm; these data are shown in Fig. 2 also and are included here for comparison), 8-p-sulfophenyltheophylline (8-pSPT; 100 μm), a charged adenosine receptor antagonist, and adenosine deaminase (ADA; 25 μg/ml). The fourth pair of bars demonstrates that including 5 mm DDA in the pipette had no significant effect on the baseline response. The last pair of bars shows that there was no effect of 2 mm BaCl₂ on these responses (compare with first set of bars). The number on eachbar indicates the number of cells recorded; *p < 0.05; **p < 0.01 relative to 0 mm adenosine.

Fig. 5.

The paired-pulse facilitation (PPF) ratio of excitatory postsynaptic currents evoked in neurons recorded with control and adenosine-containing electrode solutions. The baseline magnitude of PPF in control neurons was significantly different (p < 0.05) from that in cells loaded with 5 mm adenosine (A), and this difference was abolished during superfusion with 200 μm theophylline. Individual points represent the mean ± SEM for the control (n = 5) or adenosine-loaded (n = 6) group of cells. B, A pair of EPSCs evoked 50 msec apart in a cell recorded with no adenosine added to the pipette. C, Comparable responses in an adenosine-loaded cell (5 mm adenosine); both are averages of seven evoked responses.

Fig. 6.

The effects of 200 μm theophylline on simultaneously recorded intracellular EPSCs and extracellular field excitatory postsynaptic potentials (fEPSPs). A, The ensemble average response (mean ± SEM) induced by application of 200 μm theophylline for the EPSC recorded from control cells (•; n = 5) or 5 mmadenosine-loaded cells (○; n = 5). The duration of theophylline application is indicated by the horizontal bar at the bottom of the graph. B, The concurrent change in the fEPSP (mean ± SEM) when simultaneous whole-cell recording was conducted with control solution (•;n = 5 slices) or electrode solution containing 5 mm adenosine (○; n = 5 slices).C, D, The effect of 200 μmtheophylline on the EPSC (C) from a cell recorded with an adenosine-free electrode solution and the corresponding fEPSP responses (D). The smaller response in each case is the pre-drug control, and the larger response is during theophylline superfusion. The tracings are averages of 8–12 sweeps.E, F, Corresponding responses from a cell recorded with an electrode solution containing 5 mmadenosine (E) and the corresponding fEPSP responses (F). In both cases, the larger response was recorded in the presence of 200 μm theophylline.