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Exocytosis of ATP From Astrocytes Modulates Phasic and Tonic Inhibition in the Neocortex

Figure 5

Intracellular perfusion of astrocyte with inhibitors of vesicular transport of ATP selectively affects slower purinergic EPSCs.

Whole-cell recordings of spontaneous EPSCs in the pyramidal neurons of somatosensory cortex of wild-type mice were carried out simultaneously with intracellular perfusion of astrocyte located in close proximity. Spontaneous currents recorded in the pyramidal neurons at a holding potential of −80 mV in the presence of picrotoxin (100 µM), CNQX (50 µM), and intracellular MK801 (10 µM) were mediated by P2X receptors, as demonstrated in Figures 3 and 4; the intracellular solution contained fluorescent dye Texas Red (30 µM). Whole-cell recording configuration was also established for neighboring astrocytes 15 min prior to the start of recording in the neuron, and the holding potential was −80 mV. Intracellular solution contained either fluorescent dye AlexaFluor 488 (30 µM) alone or AlexaFluor 488 and 1 mM of diadenosine-triphosphate (AP3A) or UTP. (A) A representative two-photon image of the recording outline and green and red fluorescence images (projection of Z-stack) are merged. (B) Two episodes of 100 Hz stimulation (HFS) of cortical afferents were delivered to trigger Ca2+ transients in the astrocytes, as demonstrated in Figure 4. Each dot shows the average amplitude and frequency of spontaneous currents recorded in a 1 min time window in the pyramidal neurons during perfusion of astrocyte with AlexaFluor 488 only (contro) and AlexaFluor and AP3A; data are presented as mean ± SD for five neurons. The asterisk (*) indicates the significant difference (p<0.05, unpaired t test) from the control values. The decrease in amplitude and significant increase in frequency of purinergic sEPSCs were attenuated by perfusion of astrocyte with AP3A, and the effect was more prominent after the second HFS episode. (C) The amplitude and decay time distributions of purinergic sEPSCs recorded before and 1–3 min after the second HFS (pooled data for five neurons in each case) reveal the decrease in number of sEPSCs of smaller amplitude and slower kinetics after perfusion of astrocyte with AP3A. (D) The representative average waveforms (20 events) of fast and slow spontaneous sEPSCs (separated as shown in Figures 4 and S3) recorded 1 min after the 2-s HFS episode. Note the decrease in the amplitude of slow sEPSCs recorded in the presence of AP3A and lack of changes in the fast sEPSC. (E) Diagrams show the amplitude and frequency of slow purinergic sEPCS averaged within a 3 min time window before (baseline) and after HFS episodes delivered in control and during perfusion of astrocytes with AP3A and UTP. Data are shown as mean ± SD for the five neurons. The statistical significance of the difference from the control values was indicated as (*) p<0.05 and (**) p<0.01. Note the decrease in the mean frequency and amplitude of slow sEPSCs.

Figure 5