Exocytosis of ATP from astrocytes <i>in situ</i> can be triggered by synaptic stimulation.

<p>(A, B) Ca²⁺ signaling was monitored in astrocytes of somatosensory cortex layer 2/3 of wild-type (A) and dn-SNARE (B) mice simultaneously with voltage-clamp recordings of membrane currents in the pyramidal neurons. Spontaneous currents recorded in the pyramidal neurons at a holding potential of −80 mV in the presence of picrotoxin (100 µM) and CNQX (50 µM) were mediated by P2X receptors, as verified by inhibition with selective P2X antagonist NF279 (3 µM). The single episode of 100 Hz stimulation (HFS) triggered Ca²⁺ transients in the astrocytes of both wild-type and dn-SNARE mice; representatives are the Ca²⁺ transients and pseudo-color images (scale bar, 10 µm) recorded before (rest) and at the peak of response (stim). Ca²⁺ transients were followed by burst of spontaneous purinergic currents only in the neurons of wild-type mice (A), whereas neuronal spontaneous activity was not enhanced in the dn-SNARE mice (B). Inlays show the average waveforms (20 events) of spontaneous currents recorded before (rest) and 1 min after HFS (stim). (C, D). Each dot shows the average amplitude and frequency of spontaneous currents recorded in a 1 min time window in the pyramidal neurons of wild-type and dn-SNARE mice; data are presented as mean ± SD for six neurons. The asterisks (*) and (**) indicate the significant difference from the control values. The decrease in amplitude and significant increase in frequency of purinergic sEPSCs were observed in the wild-type but not in the dn-SNARE mice. (E, F) The amplitude and decay time distributions of purinergic sEPSCs recorded before, immediately after (0–30 s), and 1–3 min after HFS (pooled data for six neurons of each type) reveal the presence of a distinct population of spontaneous currents of smaller amplitude and slower kinetics in the wild-type but not in the dn-SNARE mice. Hence, these events are most likely activated by ATP released from astrocytes. The faster sEPSCs with larger amplitudes that underlie baseline activity both in wild-type and dn-SNARE mice are activated by ATP released from the nerve terminals. (G, H) The plot of decay time of purinergic sEPSCs against the amplitude demonstrates the presence of two populations of sEPSCs in the wild-type mice: slower currents of smaller amplitude (green area) and faster currents of higher amplitude (orange area). The corresponding waveforms (average of 20 traces) are shown in the inlay. HFS significantly increases the number of slower spontaneous currents. These currents were very rarely observed in the dn-SNARE mice, both in control and after HFS.