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Geoff Burnstock (1929-2020) discovered purinergic signaling in a fastidious research that started in early 1960 and culminated in a concept of purinergic nerves in 1972. Subsequently, Geoff developed the concept of purinergic transmission and demonstrated ATP storage, release, and degradation in the context of cotransmission, which was another fundamental concept developed by him. Purinergic transmission contributes to themost fundamental physiological functions such as sensory transduction, regulation of heart rate, smoothmuscle contraction, bile secretion, endocrine regulation, immune responses, as well as to various pathophysiological conditions, including inflammation, cancer, neuropathic pain, diabetes, and kidney failure. [ABSTRACT FROM AUTHOR]

Chronic pain is a debilitating and rather common health problem. The present shortage in analgesic drugs with a favorable spectrum but without remarkable side effects furthered the search for alternative therapeutic manipulations. Increasing evidence from both basic and clinical research on acupuncture, a main alternative therapy of traditional Chinese medicine, suggests that chronic pain is sensitive to acupuncture procedures. Clarification of the underlying mechanisms is a challenge of great theoretical and practical significance. The seminal hypothesis of Geoffrey Burnstock and the astounding findings of Maiken Nedergaard on the involvement of purinergic signaling in the beneficial effects of acupuncture fertilized the field and led to an intensification of research on acupurines. In this review, we will summarize the state-of-the-art situation and try to forecast how the field is likely to develop in the future. [ABSTRACT FROM AUTHOR]

The ATP-sensitive homomeric P2X7 receptor (P2X7R) has received particular attention as a potential drug target because of its widespread involvement in inflammatory diseases as a key regulatory element of the inflammasome complex. However, it has only recently become evident that P2X7Rs also play a pivotal role in central nervous system (CNS) pathology. There is an explosion of data indicating that genetic deletion and pharmacological blockade of P2X7Rs alter responsiveness in animal models of neurological disorders, such as stroke, neurotrauma, epilepsy, neuropathic pain, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, and Huntington's disease. Moreover, recent studies suggest that P2X7Rs regulate the pathophysiology of psychiatric disorders, including mood disorders, implicating P2X7Rs as drug targets in a variety of CNS pathology. [ABSTRACT FROM AUTHOR]

Epilepsy is one of the most common chronic diseases of the central nervous system (CNS). Treatment of epilepsy remains, however, a clinical challenge with over 30% of patients not responding to current pharmacological interventions. Complicating management of treatment, epilepsy comes with multiple comorbidities, thereby further reducing the quality of life of patients. Increasing evidence suggests purinergic signalling via extracellularly released ATP as shared pathological mechanisms across numerous brain diseases. Once released, ATP activates specific purinergic receptors, including the ionotropic P2X7 receptor (P2X7R). Among brain diseases, the P2X7R has attracted particular attention as a therapeutic target. The P2X7R is an important driver of inflammation, and its activation requires high levels of extracellular ATP to be reached under pathological conditions. Suggesting the therapeutic potential of drugs targeting the P2X7R for epilepsy, P2X7R expression increases following status epilepticus and during epilepsy, and P2X7R antagonism modulates seizure severity and epilepsy development. P2X7R antagonism has, however, also been shown to be effective in treating conditions most commonly associated with epilepsy such as psychiatric disorders and cognitive deficits, which suggests that P2X7R antagonisms may provide benefits beyond seizure control. This review summarizes the evidence suggesting drugs targeting the P2X7R as a novel treatment strategy for epilepsy with a particular focus of its potential impact on epilepsy-associated comorbidities. [ABSTRACT FROM AUTHOR]

The present study was aimed to clarify the role of purinergic signalling in the regulation of ingestion behaviour. The ATP/ADP analogues 2-methylthioATP (2-MeSATP) and adenosine 5′-O-(2-thiodiphosphate) (ADPβS) increased the food intake after intracerebroventricular infusion in 18-h food-deprived rats. This effect was abolished by pretreatment with the non-selective P2X/P2Y receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) or the selective P2Y1 receptor antagonist MRS 2179, respectively. The stimulation of food intake mediated by ADPβS was also blocked by pretreatment with the nitric oxide synthase (NOS) inhibitor Nw-nitro-l-arginine methylester (L-NAME), as well as with the inhibitor of the soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), suggesting that the orexigenic effect seems to be closely related with the ensuing formation of nitric oxide. The immunohistochemical staining indicating a co-localization of P2Y1 receptor- and nNOS-immunoreactivities in a population of neurons in the ventromedial hypothalamic nucleus (VMH) agrees with this assumption. Further experiments with the direct local application of these compounds into the VMH and lateral hypothalamic nucleus (LH) show that the stimulation of P2Y1 receptors in these functionally antagonistic brain regions exerts an increased food intake. Hence, different signal transduction mechanisms may operate in the VMH and LH. Our assumption is supported by distinct effects of the NOS inhibitor L-NAME in these two hypothalamic nuclei. The present data suggest that ATP/ADP, acting as extracellular signal molecules in the rat brain, are involved in the regulation of food intake, possibly depending on P2Y1-receptor-mediated nitric oxide production. [ABSTRACT FROM AUTHOR]

Abstract: Extracellular purines act as neuromodulators on transmitter release and may exert toxic effects at higher concentrations. In microdialysis studies, endogenous ATP facilitated the extracellular concentration of glutamate in the nucleus accumbens (NAc) of rats. Additionally, P2 receptors are involved in astrogliosis in vivo after a stab wound injury in the same region, suggesting that these receptors, preferentially the metabotropic P2Y1 receptor subtype, mediate also trophic responses. Two sets of experimental findings support the involvement of purinergic and glutamatergic mechanisms in the response of brain to mechanical damage. First, in the present studies, the initial time course of extracellular ATP and glutamate was analyzed after a mechanical injury. The concentration of ATP in microdialysates was elevated only in the first 15-min sample whereas glutamate returned to a basal concentration not before a 90-min period had elapsed. We suggest, that the acute injury-evoked stimulation of P2 receptors contributes to glutamate-mediated excitotoxicity. Second, the expression of P2Y1 receptors and their possible relation to glutamatergic structures, identified by neuronal vesicular glutamate transporters (VGLUTs), were elucidated in non-treated and mechanically injured animals after 4 days. The number of P2Y1-positive cells was significantly increased after injury. Furthermore, P2Y1 receptor-labeled cells do not exhibit immunoreactivity for VGLUT1 and VGLUT2 without and after injury. However, after injury, a co-expression of the P2Y1 receptor on VGLUT3-immunopositive cells in the NAc was observed. No VGLUT1-, 2- and 3-immunoreactivity was found on P2Y1-positive glial fibrillary acidic protein-immunopositive astrocytes at both conditions. Our data suggest that the expression of P2Y1 receptors at neurons and astrocytes is modulated in response to cerebral injury. It can be assumed, that the enhanced sensitivity of neurons to purinergic signaling may be related directly or indirectly to changes of the glutamatergic transmission. [Copyright &y& Elsevier]

Neuronally enriched primary cerebrocortical cultures were exposed to glucose-free medium saturated with argon ( in vitro ischemia) instead of oxygen (normoxia). Ischemia did not alter P2X7 receptor mRNA, although serum deprivation clearly increased it. Accordingly, P2X7 receptor immunoreactivity (IR) of microtubuline-associated protein 2 (MAP2)-IR neurons or of glial fibrillary acidic protein (GFAP)-IR astrocytes was not affected; serum deprivation augmented the P2X7 receptor IR only in the astrocytic, but not the neuronal cell population. However, ischemia markedly increased the ATP- and 2′-3′- O-(4-benzoylbenzoyl)-adenosine 5′-triphosphate (BzATP)-induced release of previously incorporated [3H]GABA. Both Brilliant Blue G and oxidized ATP inhibited the release of [3H]GABA caused by ATP application; the Brilliant Blue G-sensitive, P2X7 receptor-mediated fraction, was much larger after ischemia than after normoxia. Whereas ischemic stimulation failed to alter the amplitude of ATP- and BzATP-induced small inward currents recorded from a subset of non-pyramidal neurons, BzATP caused a more pronounced increase in the frequency of miniature inhibitory postsynaptic currents (mIPSCs) after ischemia than after normoxia. Brilliant Blue G almost abolished the effect of BzATP in normoxic neurons. Since neither the amplitude of mIPSCs nor that of the muscimol-induced inward currents was affected by BzATP, it is assumed that BzATP acts at presynaptic P2X7 receptors. Finally, P2X7 receptors did not enhance the intracellular free Ca2+ concentration either in proximal dendrites or in astrocytes, irrespective of the normoxic or ischemic pre-incubation conditions. Hence, facilitatory P2X7 receptors may be situated at the axon terminals of GABAergic non-pyramidal neurons. When compared with normoxia, ischemia appears to markedly increase P2X7 receptor-mediated GABA release, which may limit the severity of the ischemic damage. At the same time we did not find an accompanying enhancement of P2X7 mRNA or protein expression, suggesting that receptors may become hypersensitive because of an increased efficiency of their transduction pathways. [ABSTRACT FROM AUTHOR]

Patch-clamp recordings from small-diameter rat dorsal root ganglion (DRG) neurons maintained in culture demonstrated preferential inhibition by ATP of high-voltage-activated, but not low-voltage-activated, Ca&sup2+; currents (I[subCa]). The rank order of agonist potency was UTP > ADP > ATP. ATP depressed the ω-conotoxin GVIA-sensitive N-type current only. Pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and 2'-deoxy-N[sup6]-methyladenosine 3',5'-bisphosphate tetraammonium, two P2Y[sub1] receptor antagonists, almost abolished the ATP-induced inhibition. Both patch-clamp recordings and immunocytochemistry coupled with confocal laser microscopy indicated a colocalization of functional P2X[sub3] and P2Y[sub1] receptors on the same DRG neurons. Because the effect of ATP was inhibited by intracellular guanosine 5'-0-(2-thiodiphosphate) or by applying a strongly depolarizing prepulse, P2Y[sub1] receptors appear to block I[subCa] by a pathway involving the βγ subunit of a G[subq/11] protein. Less efficient buffering of the intracellular Ca&sup2+; concentration ([Ca&sup2+;][subi]) by reducing the intrapipette EGTA failed to interfere with the ATP effect. Fura-2 microfluorimetry suggested that ATP raised [Ca&sup2+;][subi] by a Gα-mediated release from intracellular pools and simultaneously depressed the high external potassium concentration-induced increase of [Ca&sup2+;][subi] by inhibiting I[subCa] via Gβγ. Adenosine 5'-0-(2-thiodiphosphate) inhibited dorsal root-evoked polysynaptic population EPSPs in the hemisected rat spinal cord and prolonged the nociceptive threshold on intrathecal application in the tail-flick assay. These effects were not antagonized by PPADS. Hence, P2Y receptor activation by ADP, which is generated by enzymatic degradation of ATP, may decrease the release of glutamate from DRG terminals in the spinal cord and thereby partly counterbalance the algogenic effected on ATP. [ABSTRACT FROM AUTHOR]

Neurons of the central nervous system (CNS) are endowed with ATP-sensitive receptors belonging to the P2X (ligand-gated cationic channels) and P2Y (G protein-coupled receptors) types. Whereas a number of P2X receptors mediate fast synaptic responses to the transmitter ATP, P2Y receptors mediate either slow changes of the membrane potential in response to non-synaptically released ATP or the interaction with receptors for other transmitters. To date seven P2X and seven P2Y receptors of human origin have been molecularly identified and functionally characterized. P2X subunits may occur as homooligomers or as heterooligomeric assemblies of more than one subunit. P2X7 subunits do not form heterooligomeric assemblies and are uniqe in mediating apoptosis and necrosis of glial cells and possibly also of neurons. The P2X2, P2X4, P2X4/P2X6 and P2Y1 receptors appear to be the predominant neuronal types. The localisation of these receptors may be at the somato-dendritic region (postsynaptic) or at the nerve terminals (presynaptic). Postsynaptic P2 receptors appear to be mostly excitatory, while presynaptic P2 receptors may be either excitatory (P2X) or inhibitory (P2Y). Since in the CNS the stimulation of a single neuron may activate multiple networks, a concomitant stimulation of facilitatory and inhibitory circuits as a result of ATP release is also possible. Finally, the enzymatic degradation of ATP may lead to the local generation of adenosine which can modulate via A1 or A2A receptor-activation the ATP effect. [Copyright &y& Elsevier]

• Acupuncture analgesia is based on ATP release from (sub)cutaneous/muscular tissue. • ATP and its degradation product adenosine participate in acupuncture analgesia. • Peripheral and central mechanisms participate in the effect of acupuncture analgesia. • A1/P2X3,4,7 receptors appear to mediate acupuncture analgesia. Purinergic signaling has recently been suggested to constitute the cellular mechanism underlying acupuncture-induced analgesia (AA). By extending the original hypothesis on endogenous opioids being released during AA, Geoffrey Burnstock and Maiken Nedergaard supplied evidence for the involvement of purinoceptors (P2 and P1/A1 receptors) in the beneficial effects of AA. In view of certain pain states (e.g. neuropathic pain) which respond only poorly to therapy with standard analgesics, as well as with respect to the numerous unwanted effects of opioids and non-steroidal anti-inflammatory drugs, it is of great significance to search for alternative therapeutic options. Because clinical studies on AA yielded sometimes heterogeneous results, it is of eminent importance to relay on experiments carried out on laboratory animals, by evaluating the data with stringent statistical methods including comparison with a sufficient number of control groups. In this review, we summarize the state of the art situation with respect to the participation of P2 receptors in AA and try to forecast how the field is likely to move forward in the future. [ABSTRACT FROM AUTHOR]

• Functional P2X7 receptors are missing at neurons of the mouse hippocampus. • Astrocytic/microglial P2X7 receptors are primary targets of ATP in mouse hippocampus. • Astrocytes and neurons cross-talk via astrocytic glutamate/GABA in mouse hippocampus. Whole-cell patch clamp recordings demonstrated that in the dentate gyrus (DG) as well as in the CA3 area of mouse hippocampal slices the prototypic P2X7 receptor (R) agonist dibenzoyl-ATP (Bz-ATP) induced inward current responses both in neurons and astrocytes. Whereas the selective P2X7R antagonist A438079 strongly inhibited both neuronal and astrocytic currents, a combination of ionotropic glutamate receptor (CNQX, AP-5) and GABA A -R (gabazine) antagonists depressed the Bz-ATP-induced current responses in the DG (granule cells) and CA3 neurons only. It was concluded that Bz-ATP activated astrocytic P2X7Rs and thereby released glutamate and GABA to stimulate nearby neurons. The residual A438079-resistant current response of astrocytes was suggested to be due to the stimulation of P2XRs of the non-P2X7-type. Further, we searched for presynaptic P2X7Rs at the axon terminals of DG and CA3 pyramidal neurons innervating CA3 and CA1 cells, respectively. Bz-ATP potentiated the frequency of spontaneous postsynaptic currents (sPSCs) in CA1 but not CA3 pyramidal cells. However, the Bz-ATP effect in CA1 cells was inhibited by gabazine or the astrocytic toxin fluorocitrate suggesting stimulation of P2X7Rs at stratum radiatum astrocytes located near to interneurons and synapsing onto CA1 neurons. Our data suggest that functional P2X7Rs are missing at neurons in the tri-synaptic network of the rodent hippocampus, but are present at nearby astrocytes indirectly regulating network activity. [ABSTRACT FROM AUTHOR]

Abstract: There is no in situ evidence hitherto for a modulation by ATP of the glutamatergic excitatory transmission onto medium spiny neurons (MSNs) in the rat striatum. In order to resolve this question, we used the patch-clamp technique in brain slice preparations to record excitatory postsynaptic currents (EPSCs) evoked by intrastriatal electrical stimulation and applied N-methyl-d-aspartate (NMDA) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) to activate transmembrane currents of MSNs. In the absence of external Mg2+, ATP caused a higher maximum inhibition of the EPSCs than adenosine. Only P1 (A1), but not P2 receptor antagonists interfered with the effects of both ATP and adenosine. Moreover, A1 receptor antagonists were less potent in blocking the inhibition by ATP than that by adenosine. Eventually, adenosine deaminase (ADA) almost abolished the adenosine-induced inhibition, but only moderately decreased the ATP-induced inhibition. Antagonists of A1 receptors (but not of P2 receptors) counteracted the depression by ATP of the current responses to exogenous NMDA, without altering those to AMPA. It is suggested that ATP indirectly, via its degradation product adenosine, stimulates presynaptic inhibitory A1 receptors situated at glutamatergic nerve terminals of striatal afferents; these nerve terminals are devoid of P2 receptors. However, ATP, in contrast to adenosine, also activates postsynaptic A1 receptors at the MSN neurons themselves. The resulting negative interaction with NMDA receptors requires localized extracellular catabolism of ATP by ectonucleotidases. [Copyright &y& Elsevier]

Abstract: Purinergic neurotransmission, involving release of ATP as an efferent neurotransmitter was first proposed in 1972. Later, ATP was recognised as a cotransmitter in peripheral nerves and more recently as a cotransmitter with glutamate, noradrenaline, GABA, acetylcholine and dopamine in the CNS. Both ATP, together with some of its enzymatic breakdown products (ADP and adenosine) and uracil nucleotides are now recognised to act via P2X ion channels and P1 and P2Y G protein-coupled receptors, which are widely expressed in the brain. They mediate both fast signalling in neurotransmission and neuromodulation and long-term (trophic) signalling in cell proliferation, differentiation and death. Purinergic signalling is prominent in neurone–glial cell interactions. In this review we discuss first the evidence implicating purinergic signalling in normal behaviour, including learning and memory, sleep and arousal, locomotor activity and exploration, feeding behaviour and mood and motivation. Then we turn to the involvement of P1 and P2 receptors in pathological brain function; firstly in trauma, ischemia and stroke, then in neurodegenerative diseases, including Alzheimer''s, Parkinson''s and Huntington''s, as well as multiple sclerosis and amyotrophic lateral sclerosis. Finally, the role of purinergic signalling in neuropsychiatric diseases (including schizophrenia), epilepsy, migraine, cognitive impairment and neuropathic pain will be considered. [Copyright &y& Elsevier]

Abstract: Long-term depression (LTD) is a form of synaptic plasticity that may contribute to information storage in the central nervous system. Here we report that LTD can be elicited in layer 5 pyramidal neurons of the rat prefrontal cortex by pairing low frequency stimulation with a modest postsynaptic depolarization. The induction of LTD required the activation of both metabotropic glutamate receptors of the mGlu1 subtype and voltage-sensitive Ca2+ channels (VSCCs) of the T/R, P/Q and N types, leading to the stimulation of intracellular inositol trisphosphate (IP3) receptors by IP3 and Ca2+. The subsequent release of Ca2+ from intracellular stores activated the protein phosphatase cascade involving calcineurin and protein phosphatase 1. The activation of purinergic P2Y1 receptors blocked LTD. This effect was prevented by P2Y1 receptor antagonists and was absent in mice lacking P2Y1 but not P2Y2 receptors. We also found that activation of P2Y1 receptors inhibits Ca2+ transients via VSCCs in the apical dendrites and spines of pyramidal neurons. In addition, we show that the release of ATP under hypoxia is able to inhibit LTD by acting on postsynaptic P2Y1 receptors. In conclusion, these data suggest that the reduction of Ca2+ influx via VSCCs caused by the activation of P2Y1 receptors by ATP is the possible mechanism for the inhibition of LTD in prefrontal cortex. [ABSTRACT FROM AUTHOR]

Abstract: Small- to medium-sized neurons in the dorsal root ganglion (DRG) convey nociceptive information to the spinal cord. The co-expression of TRPV1 receptors (sensitive to vanilloids, heat and acidic pH) with P2X3 receptors (sensitive to extracellular ATP) has been found in many DRG neurons. We investigated whether the co-activation of these two receptor classes in small-diameter cells leads to a modulation of the resulting current responses shaping the intensity of pain sensation. The whole-cell patch clamp method was used to record agonist-induced currents in cultured rat DRG neurons and in HEK293 cells transfected with the respective wild-type recombinant receptors or their mutants. Co-immunoprecipitation studies were used to demonstrate the physical association of TRPV1 and P2X3 receptors. At a negative holding potential, the P2X3 receptor agonist α,β-meATP induced less current in the presence of the TRPV1 agonist capsaicin than that in its absence. This inhibitory interaction was not changed at a positive holding potential, in a Ba2+-containing superfusion medium, or when the buffering of intrapipette Ca2+ was altered. The C-terminal truncation at Glu362 of P2X3 receptors abolished the TRPV1/P2X3 cross-talk in the HEK293 expression system. Co-immunoprecipitation studies with polyclonal antibodies generated against TRPV1 and P2X3 showed a visible signal in HEK293 cells transfected with both receptors. It is concluded that the two pain-relevant receptors TRPV1 and P2X3 interact with each other in an inhibitory manner probably by a physical association established by a motif located at the C-terminal end of the P2X3 receptor distal to Glu362. [Copyright &y& Elsevier]

Abstract: Wild-type human (h) P2X3 receptors expressed in HEK293 cells responded to the prototypic agonist α,β-methylene ATP (α,β-meATP) with rapidly desensitizing inward currents and an increase in the intracellular Ca2+ concentration. In contrast to electrophysiological recordings, Ca2+ microfluorimetry showed a lower maximum of the concentration-response curve of α,β-meATP in the transiently than in the permanently transfected HEK293 cells. However, the concentrations causing 50% of the maximum possible effect (EC50 values) were identical, when measured with either method. In order to determine the role of certain conserved, positively charged amino acids in the nucleotide binding domains (NBD-1-4) of hP2X3 receptors for agonist binding, the lysine-63, -65, -176 and -299 as well as the arginine-281 and -295 residues were substituted by the neutral amino acid alanine. We observed no effect of α,β-meATP at the K63A, K176A, R295A, and K299A mutants, and a marked decrease of agonist potency at the K65A and R281A mutants. The P2X3 receptor antagonist 2'',3''-O-trinitrophenyl-ATP (TNP-ATP) blocked the effect of α,β-meATP at the wild-type hP2X3 receptor with lower affinity than at the mutant K65A, indicating an interference of this mutation with the docking of the antagonist with its binding sites. The use of confocal fluorescence microscopy in conjunction with an antibody raised against the extracellular loop of the hP2X3 receptor documented the expression of all mutants in the plasma membrane of HEK293 cells. Eventually, we modelled the possible agonist and antagonist binding sites NBD-1-4 of the hP2X3 subunit by using structural bioinformatics. This model is in complete agreement with the available data and integrates results from mutagenesis studies with geometry optimization of the tertiary structure predictions of the receptor. [Copyright &y& Elsevier]

Abstract: P2X7 receptors are ligand-gated ion channels, expressed as homo-oligomeric assemblies of individual subunits. They are widely distributed at immunocompetent cells of the central and peripheral nervous system and are believed to be primarily involved in host-defense reaction. However, a growing amount of evidence indicates that their signaling role in the brain is more widespread than previously anticipated. In this paper, we review the present knowledge on the structural and pharmacological features of the P2X7 receptor, as well as its cell-type specific localization in the nervous system. Subsequently, the participation of P2X7 receptors in distinct neuronal, astroglial and microglial functions are described. Finally, since they may play a prominent role in certain neurologic disorders, such as ischemia-reperfusion injury, Alzheimer''s disease, spinal cord injury and sensory neuropathies, the pathological role and potential therapeutic exploitation of P2X7 receptors are also discussed. [Copyright &y& Elsevier]

ATP produces a variety of Ca2+ responses in astrocytes. To address the complex spatio-temporal Ca2+ signals, we analyzed the ATP-evoked increase in intracellular Ca2+ concentration ([Ca2+]i) in cultured rat hippocampal astrocytes using fura-2 or fluo-3 based Ca2+ imaging techniques. ATP at less than 10 nM produced elementary Ca2+ release event “puffs” in a manner independent of extracellular Ca2+. Stimulation with higher ATP concentrations (3 or 10 μM) resulted in global Ca2+ responses such as intercellular Ca2+ wave. These Ca2+ responses were mainly mediated by metabotropic P2Y receptors. ATP acting on both P2Y1 and P2Y2 receptors produced a transient Ca2+ release by inositol 1,4,5-trisphosphate (InsP3). When cells were stimulated with ATP much longer, the transient [Ca2+]i elevation was followed by sustained Ca2+ entry from the extracellular space. This sustained rise in [Ca2+]i was inhibited by Zn2+ (<10 μM), an inhibitor of capacitative Ca2+ entry (CCE). CCE induced by cyclopiazonic acid or thapsigargin and Ca2+ entry evoked by ATP share the same pharmacological profile in astrocytes. Taken together, the hierarchical Ca2+ responses to ATP were observed in hippocampal astrocytes, i.e., puffs, global Ca2+ release by InsP3, and CCE in response to depletion of InsP3-sensitive Ca2+ stores. It should be noted that these Ca2+ signals and their modulation by Zn2+ could occur in the hippocampus in situ since both ATP and Zn2+ are rich in the hippocampus and could be released by excitatory stimulation. [Copyright &y& Elsevier]