Susanna Alloisio

ATP-induced, sustained calcium signalling in cultured rat cortical astrocytes: evidence for a non-capacitative, P2X7-like-mediated calcium entry

The receptor mechanisms regulating the ATP‐induced free cytosolic Ca2+ concentration ([Ca2+]i) changes in cultured rat cortical type‐1 astrocytes were analyzed using fura‐2‐based Ca2+ imaging microscopy. Upon prolonged ATP challenge (1–100 μM), astroglial cells displayed a biphasic [Ca2+]i response consisting of an initial peak followed by a sustained elevation. Suramin and pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid blocked both components, albeit to a different extent. By contrast, the selective P2X7 antagonist oxidized ATP irreversibly abrogated the sustained [Ca2+]i signal without affecting the transient phase. Finally, astrocyte challenge with the selective P2X7 agonist 3′‐O‐(4‐benzoyl)benzoyl‐ATP evoked a sustained [Ca2+]i elevation, which occluded that induced by ATP. We can conclude that in cultured cortical astrocytes the ATP‐mediated sustained [Ca2+]i rise does not implicate capacitative Ca2+ entry but involves Ca2+ influx through P2X7‐like receptors.

Adult astroglia is competent for Na+/Ca2+ exchanger-operated exocytotic glutamate release triggered by mild depolarization.

Glutamate release induced by mild depolarization was studied in astroglial preparations from the adult rat cerebral cortex, that is acutely isolated glial sub‐cellular particles (gliosomes), cultured adult or neonatal astrocytes, and neuron‐conditioned astrocytes. K+ (15, 35 mmol/L), 4‐aminopyridine (0.1, 1 mmol/L) or veratrine (1, 10 μmol/L) increased endogenous glutamate or [3H]d‐aspartate release from gliosomes. Neurotransmitter release was partly dependent on external Ca2+, suggesting the involvement of exocytotic‐like processes, and partly because of the reversal of glutamate transporters. K+ increased gliosomal membrane potential, cytosolic Ca2+ concentration [Ca2+]i, and vesicle fusion rate. Ca2+ entry into gliosomes and glutamate release were independent from voltage‐sensitive Ca2+ channel opening; they were instead abolished by 2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]ethyl]isothiurea (KB‐R7943), suggesting a role for the Na+/Ca2+ exchanger working in reverse mode. K+ (15, 35 mmol/L) elicited increase of [Ca2+]i and Ca2+‐dependent endogenous glutamate release in adult, not in neonatal, astrocytes in culture. Glutamate release was even more marked in in vitro neuron‐conditioned adult astrocytes. As seen for gliosomes, K+‐induced Ca2+ influx and glutamate release were abolished by KB‐R7943 also in cultured adult astrocytes. To conclude, depolarization triggers in vitro glutamate exocytosis from in situ matured adult astrocytes; an aptitude grounding on Ca2+ influx driven by the Na+/Ca2+ exchanger working in the reverse mode.

P2X7 pre-synaptic receptors in adult rat cerebrocortical nerve terminals: a role in ATP-induced glutamate release.

Although growing evidence suggests that extracellular ATP might play roles in the control of astrocyte/neuron crosstalk in the CNS by acting on P2X7 receptors, it is still unclear whether neuronal functions can be attributed to P2X7 receptors. In the present paper, we investigate the location, pharmacological profile, and function of P2X7 receptors on cerebrocortical nerve terminals freshly prepared from adult rats, by measuring glutamate release and calcium accumulation. The preparation chosen (purified synaptosomes) ensures negligible contamination of non‐neuronal cells and allows exposure of ‘nude’ release‐regulating pre‐synaptic receptors. To confirm the results obtained, we also carried out specific experiments on human embryonic kidney 293 cells which had been stably transfected with rat P2X7 receptors. Together, our findings suggest that (i) P2X7 receptors are present in a subpopulation of adult rat cerebrocortical nerve terminals; (ii) P2X7 receptors are localized on glutamatergic nerve terminals; (iii) P2X7 receptors play a significant role in ATP‐evoked glutamate efflux, which involves Ca2+‐dependent vesicular release; and (iv) the P2X7 receptor itself constitutes a significant Ca2+‐independent mode of exit for glutamate.

Functional evidence for presynaptic P2X7 receptors in adult rat cerebrocortical nerve terminals

The presynaptic P2X7 receptor (P2X7R) plays an important role in the modulation of transmitter release. We recently demonstrated that, in nerve terminals of the adult rat cerebral cortex, P2X7R activation induced Ca2+‐dependent vesicular glutamate release and significant Ca2+‐independent glutamate efflux through the P2X7R itself. In the present study, we investigated the effect of the new selective P2X7R competitive antagonist 3‐(5‐(2,3‐dichlorophenyl)‐1H‐tetrazol‐1‐yl)methyl pyridine (A‐438079) on cerebrocortical terminal intracellular calcium (intrasynaptosomal calcium concentration;[Ca2+]i signals and glutamate release, and evaluated whether P2X7R immunoreactivity was consistent with these functional tests. A‐438079 inhibited functional responses. P2X7R immunoreactivity was found in about 45% of cerebrocortical terminals, including glutamatergic and non‐glutamatergic terminals. This percentage was similar to that of synaptosomes showing P2X7R‐mediated [Ca2+]i signals. These findings provide compelling evidence of functional presynaptic P2X7R in cortical nerve terminals.

Differential modulation of ATP‐induced calcium signalling by A1 and A2 adenosine receptors in cultured cortical astrocytes

Despite the accumulating evidence that under various pathological conditions the extracellular elevation of adenine‐based nucleotides and nucleosides plays a key role in the control of astroglial reactivity, how these signalling molecules interact in the regulation of astrocyte function is still largely elusive. The action of the nucleoside adenosine in the modulation of the intracellular calcium signalling ([Ca2+]i) elicited by adenosine 5′‐triphosphate (ATP)‐induced activation of P2 purinoceptors was investigated on neocortical type‐1 astrocytes in primary culture by using single‐cell microfluorimetry. Astrocyte challenge with ATP (1–10 μM) elicited biphasic [Ca2+]i responses consisting of an initial peak followed by a sustained elevation. The stable adenosine analogue 2‐chloroadenosine (2‐ClA) potentiated the transient [Ca2+]i rise induced by activation of metabotropic P2Y receptors. Among the various P1 receptor agonists tested, the nonselective agonist 5′‐N‐ethylcarboxamidoadenosine (NECA) mimicked the 2‐ClA action, whereas the selective A1 R(−) N6‐(2‐phenylisopropyl)‐adenosine (R‐PIA), the A2A 2‐[4‐(2‐carboxyethyl)phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS‐21680) and A3 1‐deoxy‐1‐(6‐[([3‐lodophenyl]methyl)‐amino]‐9H‐purin‐9‐yl)‐N‐methyl‐β‐D‐ribofuranuronamide (IB‐MECA) agonists were ineffective. Application of R‐PIA>NECA2‐ClA depressed the [Ca2+]i plateau reversibly. Moreover, in the presence of R‐PIA or 2‐ClA, the prolonged [Ca2+]i signal was maintained by application of the A1 antagonist 1,3‐diethyl‐8‐phenylxanthine (DPX). Finally, preincubation of the astrocytes with pertussis toxin abrogated the 2‐ClA inhibition of the ATP‐elicited sustained [Ca2+]i rise without affecting the transient [Ca2+]i potentiation. Taken together, these findings indicate that stimulation of A1 and A2 adenosine receptors mediates a differential modulation of [Ca2+]i signalling elicited by P2 purinoceptors. Since variations in [Ca2+]i dynamics also affect cell proliferation and differentiation, our data suggest that tuning of the extracellular levels of adenosine may be relevant for the control of astrogliosis mediated by adenine nucleotides.

Potentiation of Native and Recombinant P2X7-Mediated Calcium Signaling by Arachidonic Acid in Cultured Cortical Astrocytes and Human Embryonic Kidney 293 Cells

In the brain, arachidonic acid (AA) plays a critical role in the modulation of a broad spectrum of biological responses, including those underlying neuroinflammation. By using microfluorometry, we investigated the action of extracellular AA in the modulation of the purinoceptor P2X7-mediated elevation of [Ca2+]i in cultured neocortical type-1 astrocytes and P2X7-, P2X2-transfected human embryonic kidney (HEK) 293 cells. We report that in cultured astrocytes, AA-induced [Ca2+]i elevation is coupled to depletion of intracellular Ca2+ stores and to a sustained noncapacitative Ca2+ entry. AA also induced a robust potentiation of the astrocytic P2X7-mediated [Ca2+]i rise evoked by the selective agonist 3′-O-(4-benzoyl)benzoyl-ATP (BzATP). Pharmacological studies demonstrate that the selective P2X7 antagonists oxidized ATP and Brilliant Blue G abrogated the AA-mediated potentiation of BzATP-evoked [Ca2+]i elevation. Fluorescent dye uptake experiments showed that the AA-induced increase in [Ca2+]i was not due to a switch of the P2X7 receptor from channel to the pore mode of gating. The synergistic effect of AA and BzATP was also observed in HEK293 cells stably expressing rat and human P2X7 but not in rat P2X2. Control HEK293 cells responded to AA exposure only with a transient [Ca2+]i elevation, whereas in those expressing the P2X7 receptor, AA elicited a potentiation of the BzATP-induced [Ca2+]i rise. Together, these findings indicate that AA mediates a complex regulation of [Ca2+]i dynamics also through P2X7-mediated Ca2+ entry, suggesting that variations in AA production may be relevant to the control of both the temporal and spatial kinetics of [Ca2+]i signaling in astroglial cells.

The P2X7 receptor as a route for non‐exocytotic glutamate release: dependence on the carboxyl tail

P2X7 receptors trigger Ca2+‐dependent exocytotic glutamate release, but also function as a route for non‐exocytotic glutamate release from neurons or astrocytes. To gain an insight into the mechanisms involving the P2X7 receptor as a direct pathway for glutamate release, we compared the behavior of a full‐length rat P2X7 receptor, a truncated rat P2X7 receptor in which the carboxyl tail had been deleted, a rat P2X7 receptor with the 18‐amino acid cysteine‐rich motif of the carboxyl tail deleted, and a rat P2X2 receptor, all of which are expressed in HEK293 cells. We found that the P2X7 receptor function as a route for glutamate release was antagonized in a non‐competitive way by extracellular Mg2+, did not require the recruitment of pore‐forming molecules, and was dependent on the carboxyl tail. Indeed, the truncated P2X7 receptor and the P2X7 receptor with the deleted cysteine‐rich motif both lost their function as a pathway for glutamate release, while still evoking intracellular Ca2+ elevation. No glutamate efflux was observed through the P2X2 receptor. Notably, HEK293 cells (lacking the machinery for Ca2+‐dependent exocytosis), when transfected with P2X7 receptors, appear to be a suitable model for investigating the P2X7 receptor as a route for non‐exocytotic glutamate efflux.

Calmidazolium selectively inhibits exocytotic glutamate release evoked by P2X7 receptor activation

We previously observed that activation of presynaptic P2X7 receptors located on rat cerebrocortical nerve terminals induced the release of glutamate through different modes: the channel conformation allowing Ca(2+) entry triggered exocytotic release, while the receptor itself functioned as a permeation pathway for the non-exocytotic glutamate release. Considering that exocytotic and non-exocytotic glutamate release evoked by the activation of P2X7 receptors might play a role in the control of glutamatergic synapses, we investigated whether calmidazolium (which has been found to inhibit small cation currents through recombinant P2X7 receptors, but not organic molecule permeation) could distinguish between P2X7-related exocytotic and non-exocytotic modes of glutamate release. We found that calmidazolium inhibited the intrasynaptosomal Ca(2+) response to P2X7 receptor activation and the Ca(2+)-dependent exocytotic glutamate release from rat cerebrocortical nerve terminals, but was ineffective against the Ca(2+)-independent glutamate release. The P2X7 competitive antagonist A-438079 eliminated both exocytotic and non-exocytotic P2X7 receptor-evoked glutamate release. Selective inhibition of exocytotic glutamate release indicates that calmidazolium inhibits events dependent on the function of native rat P2X7 receptors as Ca(2+) channels, and suggests that it can be used as a tool to dissociate P2X7-evoked exocytotic from non-exocytotic glutamate release.

Multiparametric characterisation of neuronal network activity for in vitro agrochemical neurotoxicity assessment

The last few decades have seen the marketing of hundreds of new pesticide products with a forecasted expansion of the global agrochemical industry. As several pesticides directly target nervous tissue as their mechanism of toxicity, alternative methods to routine in vivo animal testing, such as the Multi Electrode Array (MEAs)-based approach, have been proposed as an in vitro tool to perform sensitive, quick and low cost neuro-toxicological screening. Here, we examined the effects of a training set of eleven active substances known to have neuronal or non-neuronal targets, contained in the most commonly used agrochemicals, on the spontaneous electrical activity of cortical neuronal networks grown on MEAs. A multiparametric characterisation of neuronal network firing and bursting was performed with the aim of investigating how this can contribute to the efficient evaluation of in vitro chemical-induced neurotoxicity. The analysis of MFR, MBR, MBD, MISI_B and % Spikes_B parameters identified four different groups of chemicals: one wherein only inhibition is observed (chlorpyrifos, deltamethrin, orysastrobin, dimoxystrobin); a second one in which all parameters, except the MISI_B, are inhibited (carbaryl, quinmerac); a third in which increases at low chemical concentration are followed by decreases at high concentration, with exception of MISI_B that only decreased (fipronil); a fourth in which no effects are observed (paraquat, glyphosate, imidacloprid, mepiquat). The overall results demonstrated that the multiparametric description of the neuronal networks activity makes MEA-based screening platform an accurate and consistent tool for the evaluation of the toxic potential of chemicals. In particular, among the bursting parameters the MISI_B was the best that correlates with potency and may help to better define chemical toxicity when MFR is affected only at relatively high concentration.

Evidence for two conductive pathways in P2X7 receptor: differences in modulation and selectivity

J. Neurochem. (2010) 113, 796–806.