Purinergic Receptors Mediate Two Distinct Glutamate Release Pathways in Hippocampal Astrocytes

The purinergic P2X7 receptor (P2X7R) can mediate glutamate release from cultured astrocytes. Using patch clamp recordings, we investigated whether P2X7Rs have the same action in hippocampal astrocytes in situ. We found that 2- and 3-O-(4-benzoylbenzoyl)ATP (BzATP), a potent, although unselective P2X7R agonist, triggers two different glutamate-mediated responses in CA1 pyramidal neurons; they are transient inward currents, which have the kinetic and pharmacological properties of previously described slow inward currents (SICs) due to Ca2+-dependent glutamate release from astrocytes, and a sustained tonic current. Although SICs were unaffected by P2X7Rs antagonists, the tonic current was inhibited, was amplified in low extracellular Ca2+, and was insensitive to glutamate transporter and hemichannel inhibitors. BzATP triggered in astrocytes a large depolarization that was inhibited by P2X7R antagonists and amplified in low Ca2+. In low Ca2+ BzATP also induced lucifer yellow uptake into a subpopulation of astrocytes and CA3 neurons. Our results demonstrate that purinergic receptors other than the P2X7R mediate glutamate release that evokes SICs, whereas activation of a receptor that has features similar to the P2X7R, mediates a sustained glutamate efflux that generates a tonic current in CA1 neurons. This sustained glutamate efflux, which is potentiated under non-physiological conditions, may have important pathological actions in the brain. The purinergic P2X7 receptor (P2X7R) can mediate glutamate release from cultured astrocytes. Using patch clamp recordings, we investigated whether P2X7Rs have the same action in hippocampal astrocytes in situ. We found that 2- and 3-O-(4-benzoylbenzoyl)ATP (BzATP), a potent, although unselective P2X7R agonist, triggers two different glutamate-mediated responses in CA1 pyramidal neurons; they are transient inward currents, which have the kinetic and pharmacological properties of previously described slow inward currents (SICs) due to Ca2+-dependent glutamate release from astrocytes, and a sustained tonic current. Although SICs were unaffected by P2X7Rs antagonists, the tonic current was inhibited, was amplified in low extracellular Ca2+, and was insensitive to glutamate transporter and hemichannel inhibitors. BzATP triggered in astrocytes a large depolarization that was inhibited by P2X7R antagonists and amplified in low Ca2+. In low Ca2+ BzATP also induced lucifer yellow uptake into a subpopulation of astrocytes and CA3 neurons. Our results demonstrate that purinergic receptors other than the P2X7R mediate glutamate release that evokes SICs, whereas activation of a receptor that has features similar to the P2X7R, mediates a sustained glutamate efflux that generates a tonic current in CA1 neurons. This sustained glutamate efflux, which is potentiated under non-physiological conditions, may have important pathological actions in the brain. Glutamate is the principal mediator of excitatory neurotransmission in the central nervous system as well as a recognized excitotoxic agent that can lead neurons and astrocytes to death when present at excessive extracellular concentrations (1.Choi D.W. Neuron. 1988; 1: 623-634Abstract Full Text PDF PubMed Scopus (4203) Google Scholar, 2.Choi E.S.H. Clegg D.O. Dev. Biol. 1990; 142: 169-177Crossref PubMed Scopus (17) Google Scholar). The ability to release this transmitter (3.Parpura V. Basarsky T.A. Liu F. Jeftinija K. Jeftinija S. Haydon P.G. Nature. 1994; 369: 744-747Crossref PubMed Scopus (1418) Google Scholar, 4.Pasti L. Volterra A. Pozzan T. Carmignoto G. J. Neurosci. 1997; 17: 7817-7830Crossref PubMed Google Scholar, 5.Bezzi P. Carmignoto G. Pasti L. Vesce S. Rossi D. Rizzini B.L. Pozzan T. Volterra A. Nature. 1998; 391: 281-285Crossref PubMed Scopus (1002) Google Scholar) hints at direct participation of astrocytes in glutamatergic neuronal transmission as well as in the excitotoxic action of glutamate. Although this latter issue remains to be proved, increasing evidence indicates that astrocyte-derived glutamate has complex actions on neurons exerting a modulatory role on synaptic transmission. For example, in the retina the release of glutamate from astrocytes modulates ganglion cell spike activity driven by light stimulation, most likely through a presynaptic action (6.Newman E.A. Zahs K.R. J. Neurosci. 1998; 18: 4022-4028Crossref PubMed Google Scholar). In the hippocampus, it modulates excitability of interneurons and potentiates inhibitory transmission (7.Kang J. Jiang L. Goldman S.A. Nedergaard M. Nat. Neurosci. 1998; 1: 683-692Crossref PubMed Scopus (694) Google Scholar, 8.Liu Q.-S. Xu Q. Arcuino G. Kang J. Nedergaard M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3172-3177Crossref PubMed Scopus (175) Google Scholar); it acts also on excitatory axon terminals of the CA1 region to increase the probability of spontaneous glutamate release (9.Fiacco T.A. McCarthy K.D. J. Neurosci. 2004; 24: 722-732Crossref PubMed Scopus (283) Google Scholar). At the same time, astrocytic glutamate exerts a direct action on hippocampal pyramidal neurons by activating extra-synaptic NMDARs 2The abbreviations used are: NMDAR, N-methyl-d-aspartate (NMDA) receptor; P2X7R, purinergic P2X7 receptor; [Ca2+]i, intracellular Ca2+ concentration; BzATP, 2- and 3-O-(4-benzoylbenzoyl)ATP; SIC, slow inward current; TTX, tetrodotoxin; LY, lucifer yellow; d-AP5, d-(-)-2-amino-5-phosphonopentanoic acid; NBQX, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide; TBOA, DL-threo-β-benzyloxyaspartate; α,β-mATP, α,β-methylene ATP; BBG, Brilliant Blue G; OxATP, adenosine 5-triphosphate-2,3-dialdehyde; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid.2The abbreviations used are: NMDAR, N-methyl-d-aspartate (NMDA) receptor; P2X7R, purinergic P2X7 receptor; [Ca2+]i, intracellular Ca2+ concentration; BzATP, 2- and 3-O-(4-benzoylbenzoyl)ATP; SIC, slow inward current; TTX, tetrodotoxin; LY, lucifer yellow; d-AP5, d-(-)-2-amino-5-phosphonopentanoic acid; NBQX, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide; TBOA, DL-threo-β-benzyloxyaspartate; α,β-mATP, α,β-methylene ATP; BBG, Brilliant Blue G; OxATP, adenosine 5-triphosphate-2,3-dialdehyde; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid. and triggering episodic, inward currents characterized by slow kinetics (SICs) (10.Angulo M.C. Kozlov A.S. Charpak S. Audinat E. J. Neurosci. 2004; 24: 6920-6927Crossref PubMed Scopus (412) Google Scholar, 11.Fellin T. Pascual O. Gobbo S. Pozzan T. Haydon P.G. Carmignoto G. Neuron. 2004; 43: 729-743Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar). Interestingly, this NMDAR response can occur synchronously in multiple CA1 neurons, raising the possibility that it may serve to promote synchrony of neuronal activity (10.Angulo M.C. Kozlov A.S. Charpak S. Audinat E. J. Neurosci. 2004; 24: 6920-6927Crossref PubMed Scopus (412) Google Scholar, 11.Fellin T. Pascual O. Gobbo S. Pozzan T. Haydon P.G. Carmignoto G. Neuron. 2004; 43: 729-743Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar). As to the mechanism of glutamate release, it is known that [Ca2+]i elevations in astrocytes can rapidly trigger the fusion with the plasma membrane of glutamate-containing vesicles (12.Kreft M. Stenovec M. Rupnik M. Grilc S. Krz̆an M. Potokar M. Pangršic T. Haydon P.G. Zorec R. Glia. 2004; 46: 437-445Crossref PubMed Scopus (148) Google Scholar, 13.Bezzi P. Gundersen V. Galbete J.L. Seifert G. Steinhäuser C. Pilati E. Volterra A. Nat. Neurosci. 2004; 7: 613-620Crossref PubMed Scopus (583) Google Scholar). Although this exocytosis-mediated pathway is most likely involved in astrocyte-to-neuron signaling under physiological conditions, other mechanisms may contribute to this process under both physiological and pathological conditions (14.Evanko D.S. Zhang Q. Zorec R. Haydon P.G. Glia. 2004; 47: 233-240Crossref PubMed Scopus (70) Google Scholar, 15.Fellin T. Carmignoto G. J. Physiol. (Lond.). 2004; 559: 3-15Crossref Scopus (219) Google Scholar). Recently, a glutamate release mechanism that involves the P2X7R has been proposed (16.Duan S. Anderson C.M. Keung E.C. Chen Y. Swanson R.A. J. Neurosci. 2003; 23: 1320-1328Crossref PubMed Google Scholar). This receptor has an established role in cellular toxicity and inflammatory processes (17.Di Virgilio F. Immunol. Today. 1995; 16: 524-528Abstract Full Text PDF PubMed Scopus (352) Google Scholar, 18.Di Virgilio F. Chiozzi P. Ferrari D. Falzoni S. Sanz J.M. Morelli A. Torboli M. Bolognesi G. Baricordi O.R. Blood. 2001; 97: 587-600Crossref PubMed Scopus (623) Google Scholar). Upon sustained activation with ATP, it can form a pore permeable to molecules of relatively large size (<900 Da), thus allowing molecules, such as glutamate, to efflux from, or enter into the cytoplasm according to their concentration gradient. In support of the role of the P2X7R, the release of glutamate from murine-cultured astrocytes triggered by purinergic receptor agonists has been described (16.Duan S. Anderson C.M. Keung E.C. Chen Y. Swanson R.A. J. Neurosci. 2003; 23: 1320-1328Crossref PubMed Google Scholar) to be (i) larger upon stimulation with BzATP than with ATP (the latter being a weak P2X7R agonist), (ii) greatly potentiated in divalent ion-free medium, i.e. a condition that favors P2X7R openings, and (iii) blocked by P2X7R antagonists. Although these results from cultured astrocytes clearly indicate that under selected experimental conditions the P2X7R allows the efflux of glutamate into the extracellular space, no such evidence exists for astrocytes in situ. On the other hand, at physiological concentrations of extracellular Ca2+, ATP is known to trigger a significant release of glutamate from astrocytes (19.Jeremic A. Jeftinija K. Stevanovic J. Glavaski A. Jeftinija S. J. Neurochem. 2001; 77: 664-675Crossref PubMed Scopus (150) Google Scholar, 20.Coco S. Calegari F. Pravettoni E. Pozzi D. Taverna E. Rosa P. Matteoli M. Verderio C. J. Biol. Chem. 2003; 278: 1354-1362Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar), which in this case depends on a classic Ca2+-dependent pathway. Indeed, by activating both ionotropic, Ca2+-permeable P2XRs, including the P2X7R and/or metabotropic P2YRs, ATP can evoke an intracellular Ca2+ increase that in turn mediates glutamate release. The aim of this study was to gain insights into the role of the purinergic receptor P2X7 in the generation of glutamate release from in situ astrocytes. Results obtained in patch clamp recordings from neurons and astrocytes of acute hippocampal slices allow us to conclude that P2X7R activation is not involved in the episodic glutamate release that triggers SICs. In contrast, a P2X7-like receptor, possibly expressed in astrocytes and neurons, contributes significantly to increasing the extracellular concentration of glutamate, in particular when extracellular Ca2+ decreases to very low levels. Slice Preparation—Transverse hippocampal slices (350–400 μm) were prepared as previously described (4.Pasti L. Volterra A. Pozzan T. Carmignoto G. J. Neurosci. 1997; 17: 7817-7830Crossref PubMed Google Scholar, 21.Edwards F.A. Konnerth A. Sakmann B. Takahashi T. Pfluegers Arch. Eur. J. Physiol. 1989; 414: 600-612Crossref PubMed Scopus (1010) Google Scholar) from Wistar rats at postnatal days 10–23. After cutting, slices were incubated at 37 °C for a recovery period of at least 1 h. Slice cutting and incubation was performed with the following physiological saline solution: 120 mm NaCl, 3.2 mm KCl, 1 mm KH2PO4, 26 mm NaHCO3, 2 mm MgCl2, 1 mm CaCl2, 2.8 mm glucose, 2 mm sodium pyruvate, and 0.6 mm ascorbic acid at pH 7.4 with 95% O2,5%CO2. Slice incubation with 300 μm OxATP for 2–3 h and with 2–4 μm BBG for either 30 min or 1 h was carried out at 37 °C. When incubated with BBG for 30 min, slices were preincubated for 30 min in saline at 37 °C. As appropriate controls in OxATP and BBG experiments, we used slices maintained at 37 °C for either 1 or 2–3 h, respectively, and the recordings from these slices was performed the same days of the recordings from BBG- or OxATP-incubated slices. Recordings were also performed from additional neurons from control slices on days different from the days in which BBG and OxATP experiments were performed. Because the mean values of SIC frequency and tonic current amplitude from the different control experiments were similar, data from these experiments were pooled together. Patch Clamp Recordings and Data Analysis—Most of the experimental procedures are similar to that described previously (11.Fellin T. Pascual O. Gobbo S. Pozzan T. Haydon P.G. Carmignoto G. Neuron. 2004; 43: 729-743Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar). In brief, during recordings slices were perfused with the following saline solution: 120 mm NaCl, 3.2 mm KCl, 1 mm KH2PO4, 26 mm NaHCO3, 2 mm CaCl2, 2.8 mm glucose, and 1 μm glycine at pH 7.4 with 95% O2,5%CO2. Low Ca2+ solution was obtained by replacing CaCl2 with EGTA (0.25 mm). Intracellular pipette solution was 145 mm potassium gluconate, 2 mm MgCl2, 5 mm EGTA, 2 mm Na2ATP, 0.2 mm NaGTP, and 10 mm HEPES to pH 7.2 with KOH. Slices were viewed with an upright Zeiss Axioscope microscope (Carl Zeiss Spa, Milan, IT) equipped with differential interference Nomarski optics, a CCD camera (COHU Inc., San Diego, CA), and a mercury light source for fluorescence excitation. Data were amplified and filtered at 1 KHz with one or two Axopatch-200B amplifiers and sampled at 5 KHz with a Digidata 1200 interface (Axon instruments, Union City, CA). The liquid junction potential at the pipette tip was -15 mV. This value should be added to all voltages to obtain the correct value of the membrane potential in whole-cell configuration. Series resistance (6 < RS < 15 megaohms) was monitored throughout each experiment, and no compensation of RS was applied. All experiments were performed at either room temperature or 35 °C and in the presence of TTX. Astrocytes were identified on the basis of both morphological and electrophysiological criteria. Astrocytes typically have small and round cell soma (diameter 6–10 μm), lack optically apparent large processes, do not fire action potentials upon application of depolarizing current pulses, and have a low input resistance (mean: 15.5 ± 1.9 megaohms, n = 24) and highly negative resting potentials (mean, -77.3 ± 0.7 mV, n = 24). All the astrocytes considered in this study exhibited a linear I-V relationship, typical of passive astrocytes (22.Matthias K. Kirchhoff F. Seifert G. Hüttmann K. Matyash M. Kettenmann H. Steinhäuser C. J. Neurosci. 2003; 23: 1750-1758Crossref PubMed Google Scholar). Neurons were voltage-clamped at -60 mV and astrocytes at -75 mV. Clampfit 8.2 (Axon instrument) and Origin 6.0 (Microcal Software, Northampton, MA) software were used for data analysis and fitting. Transient inward currents with rise time slower than 10 ms and amplitude greater than -20 pA were classified as SICs. SIC frequency, amplitude, and kinetics were measured as described previously (11.Fellin T. Pascual O. Gobbo S. Pozzan T. Haydon P.G. Carmignoto G. Neuron. 2004; 43: 729-743Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar). In pair recording experiments, the time interval between two SICs was measured as the time interval between the onset of the current in cell 1 and the onset of the current in cell 2. Background noise amplitude was calculated as the peak-to-peak amplitude. Student's t test was performed to determine statistical significance. Data are expressed as mean ± S.E. Ca2+ Imaging and Dye Uptake—Slice incubation with 10 μm Rhod-2 AM (Molecular Probes, Eugene, OR) and 0.02% pluronic for 50 min at 37 °C under mild stirring resulted in the selective loading of the Ca2+ indicator into astrocytes (23.Mulligan S.J. MacVicar B.A. Nature. 2004; 431: 195-199Crossref PubMed Scopus (698) Google Scholar). A confocal laser scanning microscope (TCS SP2 RS, Leica, Mannheim, Germany) was used for monitoring the BzATP-induced [Ca2+]i change in these cells. Slices were continuously perfused at room temperature with the same extracellular solution as used in electrophysiological recording with 0.2 mm sulfinpyrazone. The sampling rate was 2–4 s, and 8 images were averaged for each frame. Rhod-2 fluorescence was excited at 543 nm, and emitted light above 550 nm was collected. The fluorescent signal at a given time point was expressed as ΔF/F = (F1 - F0)/F0, where F0 and F1 are the values of the fluorescence in astrocytes at rest and at the given time point, respectively. No background subtraction was applied. Dye uptake experiments were carried out incubating slices for 5–15 min with 1 μm TTX, 0.5 mg/ml lucifer yellow (LY) with or without 100 μm BzATP in the same solution used in patch clamp recording experiments under mild stirring and continuous bubbling with 95% O2, 5% CO2 to maintain pH 7.4. Incubation was terminated by washing in physiological saline, and fluorescence imaging was performed within 30 min. Fluorescence images were obtained with the same Leica confocal microscope using 458-nm excitation for LY. Cells were visually identified from the differential interference contrast image, and fluorescence intensity was measured from individual cells as the average intensity of fluorescence in a region of interest corresponding to the cell soma. No background subtraction was applied. In the 8-bit scale, a value of fluorescence intensity corresponding to 50 arbitrary units (3-fold the average background noise value: 17.7 ± 0.8 arbitrary units, n = 475) was chosen as the threshold for classifying positive cells. Background noise was not different in control slices and in slices incubated with BzATP or BzATP and low Ca2+. Because of possible damage caused by slice cutting procedures, cells located close to the surface may unspecifically take up the dye. Therefore, cells positively loaded with the dye were considered only when they were located below the first 10–15 μm from the surface. Drugs—D-AP5, NBQX, 2-methyl-6-(phenylethynyl)pyridine hydrochloride, LY367385 ((S)-(+)- α-amino-4-carboxy-2-methyl-benzene-acetic acid), TBOA, and TTX were purchased from Tocris Cookson (Buckhurst Hill, UK), and BzATP, α,β-mATP, BBG, OxATP, LY, and carbenoxolone were from Sigma. All chemicals were dissolved in water or Me2SO and then diluted in the recording physiological solution just before use. Purinergic Receptor Stimulation Evokes Transient and Sustained Activation of Neuronal NMDA Receptors—In hippocampal slices in the absence of extracellular Mg2+ and in the presence of 1 μm TTX, BzATP (100 μm), a potent although unselective agonist of P2X7R (24.North R.A. Surprenant A. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 563-580Crossref PubMed Scopus (612) Google Scholar, 25.Lambrecht G. Naunyn-Schmiedebergs Arch. Pharmacol. 2000; 362: 340-350Crossref PubMed Scopus (157) Google Scholar), evokes a complex response in 20 of 39 (51%) CA1 pyramidal neurons consisting of episodic, transient inward currents and a slowly developing tonic current (Fig. 1, A and A1). Transient inward currents had a low frequency and could occur also spontaneously (Fig. 1B). The tonic current initiated 1–2 min after the onset of the BzATP application had a mean amplitude of -80 ± 23 pA and a mean time to peak of 98 ± 9 s, and it was always associated with a more than 2-fold increase in the background noise of the current trace (n = 13, Fig. 1C). Both transient currents and the tonic current were detected in 6 of the 20 (30%) responsive neurons (Fig. 1, A and A1), whereas either the tonic current or transient currents were observed in 7 (35%) and 7 (35%) of the responsive neurons, respectively (Fig. 1, D and E). Both the transient currents (Fig. 2A) and the tonic current associated with noise increase (Fig. 2, B and C) evoked by BzATP were reversibly blocked by the NMDAR antagonist D-AP5 (50–100 μm) and can, thus, be attributed to the activation of NMDARs. These results clearly indicate that slice perfusion with BzATP triggers the release of glutamate that evokes in CA1 pyramidal neurons both transient and sustained activation of the NMDARs. BzATP-induced noise increase is not due to an increase in the spontaneous synaptic release of glutamate since in the presence of D-AP5 and TTX, the frequency and amplitude of AMPA-mediated miniature currents under basal conditions and upon BzATP stimulation were not significantly changed (12 ± 9 versus 15 ± 13 events/min, n = 4; -7.7 ± 0.2 pA, n = 115, versus -8.1 ± 0.3 pA, n = 176; p > 0.2). Glutamate Release from Astrocytes Mediates the Transient Neuronal Response—Elevations in the [Ca2+]i evoked in hippocampal CA1 astrocytes by various stimuli, including purinergic receptor agonists, have been recently shown to trigger a pulsatile release of glutamate from these cells (11.Fellin T. Pascual O. Gobbo S. Pozzan T. Haydon P.G. Carmignoto G. Neuron. 2004; 43: 729-743Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar). Glutamate released from activated astrocytes acts primarily on extra-synaptic NMDARs of CA1 pyramidal neurons to trigger episodic inward currents with characteristic slow kinetics that we have called SICs (11.Fellin T. Pascual O. Gobbo S. Pozzan T. Haydon P.G. Carmignoto G. Neuron. 2004; 43: 729-743Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar). The transient, slow events evoked by BzATP in CA1 pyramidal neurons described above are mediated exclusively by NMDARs and are insensitive to 1 μm TTX. Therefore, they have the same pharmacological profile of SICs (11.Fellin T. Pascual O. Gobbo S. Pozzan T. Haydon P.G. Carmignoto G. Neuron. 2004; 43: 729-743Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar) as well as of the slow currents described by Angulo et al. (10.Angulo M.C. Kozlov A.S. Charpak S. Audinat E. J. Neurosci. 2004; 24: 6920-6927Crossref PubMed Scopus (412) Google Scholar). As shown in Fig. 3A, the mean rise time of BzATP-induced transient currents is 70.1 ± 7.9 ms (n = 54), one order of magnitude larger than that of excitatory postsynaptic currents evoked in the same neurons by Schaffer collateral stimulation (rise time, 6.7 ± 0.5 ms, n = 22). The mean decay time was 376.5 ± 24.4 ms (Fig. 3B; n = 54), which is slower than that of the excitatory postsynaptic currents from the same neurons (τ1 = 17.9 ± 6.7 ms, τ2 = 195.5 ± 14.7 ms, n = 22). The amplitude of these events can reach several hundred pA, with a mean of -115.9 ± 15.6 pA (Fig. 3C; n = 54). These features are also typical of SICs, providing further evidence for the classification of BzATP-evoked transient currents as SICs. An additional feature of SICs is that they occur with a high degree of synchrony in different pyramidal neurons (10.Angulo M.C. Kozlov A.S. Charpak S. Audinat E. J. Neurosci. 2004; 24: 6920-6927Crossref PubMed Scopus (412) Google Scholar, 11.Fellin T. Pascual O. Gobbo S. Pozzan T. Haydon P.G. Carmignoto G. Neuron. 2004; 43: 729-743Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar). We investigated this issue by using patch clamp pair recordings from two pyramidal neurons. In two of the four pairs that displayed the transient currents upon BzATP stimulation, synchronous events were observed (Fig. 3D). The inter-event time interval histogram (Fig. 3E) shows that, similar to SICs, 42% of these events (40 of 96) are synchronized in a time window of 200 ms. Taken together, these data suggest that the BzATP-induced transient currents have the same pharmacological and biophysical properties of SICs, i.e. the NMDAR-mediated, slow inward currents that are triggered in CA1 pyramidal neurons by glutamate released from activated astrocytes. SIC and Tonic Current Generation Is Dependent on the Activation of Different Purinergic Receptors—Given that BzATP is known to be a strong agonist of the P2X7R, we next investigated the possible involvement of this receptor in the generation of the BzATP-induced SICs and tonic current. To this aim, we used OxATP (300 μm) or BBG (2–4 μm), which efficiently block the P2X7R (24.North R.A. Surprenant A. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 563-580Crossref PubMed Scopus (612) Google Scholar, 26.Jiang L.-H. Mackenzie A.B. North R.A. Surprenant A. Mol. Pharmacol. 2000; 58: 82-88Crossref PubMed Scopus (345) Google Scholar, 27.Murgia M. Hanau S. Pizzo P. Rippa M. Di Virgilio F. J. Biol. Chem. 1993; 268: 8199-8203Abstract Full Text PDF PubMed Google Scholar), although they probably also affect other P2XRs. In the presence of OxATP or BBG, BzATP triggered SICs (Fig. 4A) in 7 of 18 (39%) and in 8 of 19 (42%) neurons, respectively. The percentage of responsive neurons, the frequency of the BzATP-induced SICs, and their amplitude and kinetics were not significantly different from controls (Fig. 4B; Table 1). Interestingly, under these conditions and in all the neurons tested, BzATP failed to trigger a tonic current (Fig. 4C) or an increase in background noise (Fig. 4D). These data demonstrate that, on the one hand, SICs generation is not mediated by P2X7 receptors and, on the other, that the generation of the tonic current is dependent upon the activation of a purinergic receptor type that is blocked by OxATP and BBG.TABLE 1Amplitude and kinetics of SICs are independent of P2X7R activation Mean ± S.E., and range for the amplitude and rise and decay time of SICs evoked by BzATP or α,β-mATP under the various experimental conditions are shown.AmplitudeRise timeDecay timenpAmsmsBzATP-115.9 ± 15.670.1 ± 7.9376.5 ± 24.454(-23.0 to -496.2)(10.2 to 334.6)(42.9 to 1412.5)BzATP + BBG-94.9 ± 9.8ap > 0.153.3 ± 8.0ap > 0.1347.5 ± 48.4bp > 0.547(-20.1 to -260.0)(11.0 to 298.6)(63.1 to 1546.4)BzATP + OxATP-98.5 ± 15.2ap > 0.162.4 ± 8.4bp > 0.5318.6 ± 50.3ap > 0.134(-23.0 to -342.9)(16.8 to 256.0)(74.9 to 1398.8)α,β-mATP-109.6 ± 31.1bp > 0.570.4 ± 11.9bp > 0.5449.3 ± 53.4ap > 0.119(-23.5 to -636.2)(13.8 to 184.2)(184.3 to 933.6)BzATP + 0 Ca2+-216.7 ± 57.8cp < 0.0586.1 ± 18.4ap > 0.1490.4 ± 108.8ap > 0.113(-21.0 to -695.2)(13.1 to 206.4)(61.9 to 1327.5)a p > 0.1b p > 0.5c p < 0.05 Open table in a new tab Support for this hypothesis derives from the observation that other stimuli used to trigger SICs, such as (S)-3,5-dihydroxyphenylglycine, prostaglandin E2, or photolysis of caged-Ca2+, did not evoke a tonic current associated with an increase in background noise (11.Fellin T. Pascual O. Gobbo S. Pozzan T. Haydon P.G. Carmignoto G. Neuron. 2004; 43: 729-743Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar). Interestingly, 100 μm α,β-mATP, a purinergic agonist different from BzATP (24.North R.A. Surprenant A. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 563-580Crossref PubMed Scopus (612) Google Scholar, 25.Lambrecht G. Naunyn-Schmiedebergs Arch. Pharmacol. 2000; 362: 340-350Crossref PubMed Scopus (157) Google Scholar), also failed to trigger either a tonic current (Fig. 5, A and B; n = 13) or an increase in background noise (Fig. 5C). Conversely, α,β-mATP triggered SICs in 6 of 13 pyramidal neurons (46%) with frequency, amplitude, and kinetics similar to those of SICs evoked by BzATP (Fig. 5D; Table 1). We also investigated the hypothesis that the action of Bz-ATP on neurons is mediated by adenosine receptors that can be activated by Bz-adenosine, a product of Bz-ATP degradation by ectonucleotidase enzymes. Results obtained show that application of 100 μm adenosine always failed to trigger a tonic current in CA1 neurons (n = 13, data not shown). As previously reported (28.Gerber U. Greene R.W. Haas H.L. Stevens D.R. J. Physiol. (Lond.). 1989; 417: 567-578Crossref Scopus (140) Google Scholar), we rather observed an outward current of a mean amplitude of 41.9 ± 4.5 pA. To gain further insights into the possible involvement of the P2X7R in the generation of the tonic current, we applied BzATP in the presence of low extracellular Ca2+, a condition that results in a large increase in the P2X7R conductance (29.North R.A. Physiol. Rev. 2002; 82: 1013-1067Crossref PubMed Scopus (2470) Google Scholar). To reduce the concentration of Ca2+ in the extracellular space, we perfused slices with saline containing 0.25 mm EGTA and no added Ca2+. Noteworthy is that 20 min after the onset of slice perfusion with this saline, a depolarizing stimulus (60 mm K+) still elicited [Ca2+]i elevations in neurons (data not shown). Thus, we confirm previous observations (30.Burgo A. Carmignoto G. Pizzo P. Pozzan T. Fasolato C. J. Physiol. (Lond.). 2003; 549: 537-552Crossref Scopus (15) Google Scholar) that the reduction of extracellular Ca2+ in slices is a slow process, and after perfusion with a saline containing 0 mm Ca2+ and 0.25 mm EGTA, sufficient Ca2+ remains in the extracellular space to elicit [Ca2+]i elevations through voltage-gated Ca2+ channels. In 7 neurons in which BzATP triggered either a clear (Fig. 6A, top, n = 3) or a nearly undetectable tonic current (Fig. 6A1, top, n = 4) when a second BzATP challenge was performed in low extracellular Ca2+, the amplitude of the tonic current was drastically increased (Fig. 6, A and A1, bottom, and B, left). The increase in background noise, which is always associated with the tonic current, was similarly enhanced (Fig. 6B, right). In contrast, the kinetic features of SICs evoked by BzATP in low Ca2+ are unchanged with respect to SICs evoked by BzATP in normal Ca2+ (Table 1). Because of the fact that BzATP in low Ca2+ triggered a large increase in the background noise of the trace, SICs of small amplitude were lost in the noise, and thus, only SICs of larger amplitude could be analyzed. As such, the mean amplitude of SICs evoked by BzATP in low Ca2+ results are higher than the mean amplitude of SICs evoked by BzATP in normal Ca2+. Slice perfusion for 3–4 min with low Ca2+ in the absenc