300 MRS 2179 NTPDase OGD PPADS PS PV4 RB2 SCH 58261 ZM 241385 2′-Deoxy-N6-methyladenosine 3′,5′- bisphosphate Nucleoside triphosphate diphosphohydrolase Oxygen/glucose deprivation Pyridoxalphosphate-6-azophenyl-2′,4′- disulfonic acid Population spike Hexapotassium dihydrogen monotitanoundecatungstocobaltate(II) tridecahydrate, K6H2[TiW11CoO40]·13H2O Reactive blue 2 5-Amino-7-(2-phenylethyl)-2-(2-furyl)- pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine 4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a] [1,3,5]triazin-5-yl-amino]ethyl)phenol Historical overview The physiological roles of adenosine triphosphate (ATP) and its metabolite adenosine have been historically linked to cell metabolism since ATP is an ubiquitous intracellular energy source in a number of enzymatic processes. However, over the years both metabolites have emerged as very versatile molecules of biological systems, being implicated in a variety of cell processes, from platelet aggregation to neurotransmis- sion. The term ‘purinergic signalling’ was first introduced in the scientific literature by Burnstock [1]. An implicit concept for sustaining the hypothesis of purinergic neurotransmission proposed in the 1970s was the existence of purinergic recep- tors. The first evidence in this direction suggested the exist- ence of two different subfamilies of such ‘purinoceptors’, identified as P1 and P2 receptors, selective for adenosine and ATP respectively [2]. Four different subtypes of P1-G-protein- coupled receptors: A1, A2A, A2B and A3 are known at present [3], whereas P2 purinoceptors belong to two major families: P2X ligand-gated ion channel receptors and P2Y G-protein- coupled receptors [4]. Cloning experiments supported this classification and helped to subdivide P2 receptors into seven P2X and eight P2Y subtypes [5] (plus the recently deorphan- ized GPR17 receptor [6]). Roles of ATP in neurotransmission under normoxic conditions The first studies on the role of extracellular ATP in hippo- campal neurotransmission indicated prominent inhibitory action of this purinergic nucleotide on synaptic activity [7, 8]. However, several lines of evidence led to the hypothesis that ATP-mediated inhibition of hippocampal neurotransmis- sion was probably mediated by adenosine acting on A1 Purinergic Signalling (2007) 3:299–310 receptors. In fact, this response was theophylline-sensitive [7–9] and absent in A1 knockout mice [10]. This assumption was supported by the fact that, in the hippocampus, extracellular ATP is rapidly converted into adenosine by ecto-ATPases and ecto-nucleotidases [11] and that adenosine acting on A1 receptors exerts a well-described inhibitory role on CA1 excitatory neurotransmission [3]. In recent years, a more detailed observation of ATP-evoked effects, supported by the synthesis of new pharmacological tools, has helped to clarify the effective role of purinergic nucleotides in the hippocampus. In particular, it emerged that ATP mediates inhibitory effects by P2 receptor activation [12–14]. In a recent paper, we contributed to elucidating the role of ATP on CA1 hippocampal neurotransmission, and we reinforced the concept that ATP-mediated effects are not necessarily linked to adenosine formation in this brain region [15]. ATP is hydrolysed by ecto-NTPDase, enzymes located on the cell surface in the CNS that limit ATP, ADP and AMP spatio-temporal activity [11, 16]. There are three different known NTPDases: NTPDase1 hydrolyzes ATP and ADP equally well, NTPDase2 has a high preference for ATP, NTPDase3 is a functional intermediate, preferably hydrolyzing ATP [17]. In our study, we took advantage of some recently synthesized compounds: ARL 67156, which, at micromolar concentrations, inhibits rat NTPDase1 and 3 transiently transfected in Chinese hamster ovary cells, showing negligible activity on NTPDase2 [18]; BGO 136, a new inhibitor described as a selective NTPDase1 and 2 blocker with Ki values in the high micromolar range [19]; and the recently synthesized PV4, which strongly inhibits rat NTPDase1, 2 and 3 with Ki values in the nanomolar range [20]. By using these inhibitors that are able to block NTPDase activity without interfering with P2 receptor activation, we demonstrated that ATP exerts an inhibitory action, independent of its degradation to adenosine. As shown in Fig. 1, the application of ATP during a NTPDase activity blockade still elicits a decrease in evoked synaptic responses which is even more pronounced than that evoked by ATP alone. An excitatory effect of exogenous ATP on hippocampal neurotransmission has also been reported by different authors. This effect, which was observed after drug removal, was described to persist in in vitro preparations for a relatively prolonged period, up to 1 h [21–25]. This ‘long-lasting’ potentiation of synaptic responses was compared to electri- cally evoked LTP, firstly described in the hippocampus by Abrams and Kandel [26] and called ‘ATP-induced LTP’. In agreement, we observed a potentiation of the synaptic re- sponses after drug removal when slices were superfused in the presence of the metabolically stable ATP-analogue ATPγS (Fig. 2) [15]. In addition to this ‘long-lasting’ excitatory effect evoked by the exogenous application of P2 agonists, we also demonstrated an excitatory tone exerted by endogenous ATP.