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Microglial cells, the resident macrophages of the central nervous system (CNS), exist in a process-bearing, ramified/surveying phenotype under resting conditions. Upon activation by cell-damaging factors, they get transformed into an amoeboid phenotype releasing various cell products including pro-inflammatory cytokines, chemokines, proteases, reactive oxygen/nitrogen species, and the excytotoxic ATP and glutamate. In addition, they engulf pathogenic bacteria or cell debris and phagocytose them. However, already resting/surveying microglia have a number of important physiological functions in the CNS; for example, they shield small disruptions of the blood-brain barrier by their processes, dynamically interact with synaptic structures, and clear surplus synapses during development. In neurodegenerative illnesses, they aggravate the original disease by a microglia-based compulsory neuroinflammatory reaction. Therefore, the blockade of this reaction improves the outcome of Alzheimer's Disease, Parkinson's Disease, multiple sclerosis, amyotrophic lateral sclerosis, etc. The function of microglia is regulated by a whole array of purinergic receptors classified as P2Y12, P2Y6, P2Y4, P2X4, P2X7, A2A, and A3, as targets of endogenous ATP, ADP, or adenosine. ATP is sequentially degraded by the ecto-nucleotidases and 5'-nucleotidase enzymes to the almost inactive inosine as an end product. The appropriate selective agonists/antagonists for purinergic receptors as well as the respective enzyme inhibitors may profoundly interfere with microglial functions and reconstitute the homeostasis of the CNS disturbed by neuroinflammation., Competing Interests: All authors declare no conflicts of interest.

It is broadly recognized that ATP not only supports energy storage within cells but is also a transmitter/signaling molecule that serves intercellular communication. Whereas the fast (co)transmitter function of ATP in the peripheral nervous system has been convincingly documented, in the central nervous system (CNS) ATP appears to be primarily a slow transmitter/modulator. Data discussed in the present review suggest that the slow modulatory effects of ATP arise as a result of its vesicular/nonvesicular release from astrocytes. ATP acts together with other glial signaling molecules such as cytokines, chemokines, and free radicals to modulate neuronal circuits. Hence, astrocytes are positioned at the crossroads of the neuron-glia-neuron communication pathway., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

P2X7 receptors (Rs) mediate apoptosis/necrosis in neuronal and non-neuronal systems. Patch-clamp recordings from dentate gyrus (DG) granule cells in acutely prepared hippocampal slices of mice showed that incubation with 4-aminopyridine (4-AP) causes an excitability increase. This led to an enhanced sensitivity of P2X7Rs of the underlying subgranular zone neural progenitor cells (NPCs) towards dibenzoyl-ATP (Bz-ATP). The glutamatergic agonists NMDA and AMPA, as well as the purinergic agonist ATP also increased the Bz-ATP-induced current amplitudes (I BzATP ). Tetrodotoxin as well as the standard antiepileptic drugs phenytoin, valproic acid and gabapentin counteracted the effect of 4-AP, most likely by decreasing the firing rate and/or action potential duration of DG granule cells and in consequence the release of ATP/glutamate onto NPCs. Experiments with organotypic hippocampal slice cultures confirmed these results also under conditions when 4-AP was applied for longer time periods and at much lower concentrations than used in acute slices. It was concluded that pathological firing modelled by 4-AP might trigger a sensitivity increase of P2X7Rs leading to necrosis/apoptosis of NPCs with the subsequent decrease of NPC, and in consequence, granule cell number. Hence, supersensitive P2X7Rs may exert a beneficial counter-regulatory effect by reducing the chances for the evolution of chronic temporal lobe epilepsy by ectopically located granule cells., (Copyright © 2018. Published by Elsevier Ltd.)

P2X7 receptors (Rs) constitute a subclass of ATP-sensitive ionotropic receptors (P2X1-P2X7). P2X7Rs have many distinguishing features, mostly based on their long intracellular C terminus regulating trafficking to the cell membrane, protein-protein interactions, and post-translational modification. Their C-terminal tail is especially important in enabling the transition from the nonselective ion channel mode to a membrane pore allowing the passage of large molecules. There is an ongoing dispute on the existence of neuronal P2X7Rs with consequences for our knowledge on their involvement in neuroinflammation, aggravating stroke, temporal lobe epilepsy, neuropathic pain, and various neurodegenerative diseases. Whereas early results appeared to support the operation of P2X7Rs at neurons, more recently glial P2X7Rs are increasingly considered as indirect causes of neuronal effects. Specific tools for P2X7Rs are of limited value because of the poor selectivity of agonists, and the inherent failure of antibodies to differentiate between the large number of active and inactive splice variants, or gain-of-function and loss-of-function small nucleotide polymorphisms of the receptor. Unfortunately, the available P2RX7 knock-out mice generated by pharmaceutical companies possess certain splice variants, which evade inactivation. In view of the recently discovered bidirectional dialogue between astrocytes and neurons (and even microglia and neurons), we offer an alternative explanation for previous data, which assumedly support the existence of P2X7Rs at neurons. We think that the unbiased reader will follow our argumentation on astrocytic or microglial P2X7Rs being the primary targets of pathologically high extracellular ATP concentrations, although a neuronal localization of these receptors cannot be fully excluded either., Competing Interests: The authors declare no competing financial interests., (Copyright © 2017 the authors 0270-6474/17/377049-14$15.00/0.)

Neuroglia, represented by astrocytes, oligodendrocytes, NG glia and microglia are homeostatic, myelinating and defensive cells of the brain. Neuroglial cells express various combinations of purinoceptors, which contribute to multiple intercellular signalling pathways in the healthy and diseased nervous system. Neurological diseases are invariably associated with profound neuroglial remodelling, which is manifest by reactive gliosis, pathological remodelling and functional atrophy of various types of glial cells. Gliopathology is disease and region specific and produces multiple glial phenotypes that may be neuroprotective or neurotoxic. In this review we summarise recent knowledge on the role of glial purinergic signalling in cognitive-related neurological diseases. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Glutamate is the main excitatory neurotransmitter of the central nervous system (CNS), released both from neurons and glial cells. Acting via ionotropic (NMDA, AMPA, kainate) and metabotropic glutamate receptors, it is critically involved in essential regulatory functions. Disturbances of glutamatergic neurotransmission can be detected in cognitive and neurodegenerative disorders. This paper summarizes the present knowledge on the modulation of glutamate-mediated responses in the CNS. Emphasis will be put on NMDA receptor channels, which are essential executive and integrative elements of the glutamatergic system. This receptor is crucial for proper functioning of neuronal circuits; its hypofunction or overactivation can result in neuronal disturbances and neurotoxicity. Somewhat surprisingly, NMDA receptors are not widely targeted by pharmacotherapy in clinics; their robust activation or inhibition seems to be desirable only in exceptional cases. However, their fine-tuning might provide a promising manipulation to optimize the activity of the glutamatergic system and to restore proper CNS function. This orchestration utilizes several neuromodulators. Besides the classical ones such as dopamine, novel candidates emerged in the last two decades. The purinergic system is a promising possibility to optimize the activity of the glutamatergic system. It exerts not only direct and indirect influences on NMDA receptors but, by modulating glutamatergic transmission, also plays an important role in glia-neuron communication. These purinergic functions will be illustrated mostly by depicting the modulatory role of the purinergic system on glutamatergic transmission in the prefrontal cortex, a CNS area important for attention, memory and learning.

The substantia gelatinosa (SG) of the spinal cord processes incoming painful information to ascending projection neurons. Whole-cell patch clamp recordings from SG spinal cord slices documented that in a low Ca(2+) /no Mg(2+) (low X(2+) ) external medium adenosine triphosphate (ATP)/dibenzoyl-ATP, Bz-ATP) caused inward current responses, much larger in amplitude than those recorded in a normal X(2+) -containing bath medium. The effect of Bz-ATP was antagonized by the selective P2X7 receptor antagonist A-438079. Neuronal, but not astrocytic Bz-ATP currents were strongly inhibited by a combination of the ionotropic glutamate receptor antagonists AP-5 and CNQX. In fact, all neurons and some astrocytes responded to NMDA, AMPA, and muscimol with inward current, demonstrating the presence of the respective receptors. The reactive oxygen species H2 O2 potentiated the effect of Bz-ATP at neurons but not at astrocytes. Hippocampal CA1 neurons exhibited a behavior similar to, but not identical with SG neurons. Although a combination of AP-5 and CNQX almost abolished the effect of Bz-ATP, H2 O2 was inactive. A Bz-ATP-dependent and A-438079-antagonizable reactive oxygen species production in SG slices was proven by a microelectrode biosensor. Immunohistochemical investigations showed the colocalization of P2X7-immunoreactivity with microglial (Iba1), but not astrocytic (GFAP, S100β) or neuronal (MAP2) markers in the SG. It is concluded that SG astrocytes possess P2X7 receptors; their activation leads to the release of glutamate, which via NMDA- and AMPA receptor stimulation induces cationic current in the neighboring neurons. P2X7 receptors have a very low density under resting conditions but become functionally upregulated under pathological conditions., (© 2014 Wiley Periodicals, Inc.)

P2X7 receptors have been suggested to be located both on neurons and astrocytes of the central and peripheral nervous systems. In the present Ca(2+)-imaging and patch-clamp study, we reinvestigated these findings on mixed neuronal-astrocytic cell cultures prepared from embryonic or newborn rat hippocampi. We found in a Mg(2+)-free bath medium that the prototypic P2X7 receptor agonist dibenzoyl-adenosine triphosphate (Bz-ATP) increased the intracellular Ca(2+) concentration ([Ca(2+)]i) both in the neuronal cell bodies and in their axo-dendritic processes only to a very minor extent. However, Bz-ATP produced marked [Ca(2+)]i transients in the neuronal processes, when they grew above a glial carpet, which was uniformly sensitive to Bz-ATP. These glial signals might be misinterpreted as neuronal responses because of the poor focal discrimination by a fluorescent microscope. Most astrocytes had a polygonal shape without clearly circumscribable boundaries, but a subgroup of them had neuron-like appearance. The cellular processes of this astrocytic subgroup, just as their cell somata and their polygonal counterparts, appeared to possess a high density of functional P2X7 receptors. In contrast to astrocytes, in a low Ca(2+)/no Mg(2+)-containing bath medium, hippocampal neurons failed to respond to Bz-ATP with membrane currents. In addition, neither the amplitude nor the frequency of spontaneous excitatory postsynaptic currents, representing the quantal release of glutamate, was modified by Bz-ATP. We conclude that cultured hippocampal neurons, in contrast to astrocytes, possess P2X7 receptors, if at all, only at a low density.

CNS reparative-medicine therapeutic strategies need answers on the putative recapitulation of the basic rules leading to mammalian CNS development. To achieve this aim, we focus on the regeneration of functional connections in the mesocorticolimbic dopaminergic system. We used organotypic slice cocultures of ventral tegmental area/substantia nigra (VTA/SN) and prefrontal cortex (PFC) on a multielectrode array (MEA) platform to record spikes and local field potentials. The spontaneously growing synaptically based bidirectional bursting activity was followed from 2 to 28 days in vitro (DIV). A statistical analysis of excitatory and inhibitory neurons properties of the physiological firing activity demonstrated a remarkable, exponentially increasing maturation with a time constant of about 5-7 DIV. Immunohistochemistry demonstrated that the ratio of excitatory/inhibitory neurons (3:1) was in line with the functional results obtained. Exemplary pharmacology suggested that GABAA receptors were able to exert phasic and tonic inhibition typical of an adulthood network. Moreover, dopamine D2 receptor inactivation was equally inhibitory both on the spontaneous neuronal activity recorded by MEA and on patch-clamp electrophysiology in PFC pyramidal neurons. These results demonstrate that axon growth cones reach synaptic targets up to full functionality and that organotypic cocultures of the VTA/SN-PFC perfectly model their newly born dopaminergic, glutamatergic and GABAergic neuronal circuitries.

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 Mg(2+), ATP caused a higher maximum inhibition of the EPSCs than adenosine. Only P1 (A(1)), but not P2 receptor antagonists interfered with the effects of both ATP and adenosine. Moreover, A(1) 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 A(1) 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 A(1) 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 A(1) receptors at the MSN neurons themselves. The resulting negative interaction with NMDA receptors requires localized extracellular catabolism of ATP by ectonucleotidases., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

Purine and pyrimidine nucleotides are extracellular signaling molecules in the central nervous system (CNS) leaving the intracellular space of various CNS cell types via nonexocytotic mechanisms. In addition, ATP is a neuro-and gliotransmitter released by exocytosis from neurons and neuroglia. These nucleotides activate P2 receptors of the P2X (ligand-gated cationic channels) and P2Y (G protein-coupled receptors) types. In mammalians, seven P2X and eight P2Y receptor subunits occur; three P2X subtypes form homomeric or heteromeric P2X receptors. P2Y subtypes may also hetero-oligomerize with each other as well as with other G protein-coupled receptors. P2X receptors are able to physically associate with various types of ligand-gated ion channels and thereby to interact with them. The P2 receptor homomers or heteromers exhibit specific sensitivities against pharmacological ligands and have preferential functional roles. They may be situated at both presynaptic (nerve terminals) and postsynaptic (somatodendritic) sites of neurons, where they modulate either transmitter release or the postsynaptic sensitivity to neurotransmitters. P2 receptors exist at neuroglia (e.g., astrocytes, oligodendrocytes) and microglia in the CNS. The neuroglial P2 receptors subserve the neuron-glia cross talk especially via their end-feets projecting to neighboring synapses. In addition, glial networks are able to communicate through coordinated oscillations of their intracellular Ca(2+) over considerable distances. P2 receptors are involved in the physiological regulation of CNS functions as well as in its pathophysiological dysregulation. Normal (motivation, reward, embryonic and postnatal development, neuroregeneration) and abnormal regulatory mechanisms (pain, neuroinflammation, neurodegeneration, epilepsy) are important examples for the significance of P2 receptor-mediated/modulated processes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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 Ca(2+) channels (VSCCs) of the T/R, P/Q and N types, leading to the stimulation of intracellular inositol trisphosphate (IP3) receptors by IP3 and Ca(2+). The subsequent release of Ca(2+) from intracellular stores activated the protein phosphatase cascade involving calcineurin and protein phosphatase 1. The activation of purinergic P2Y(1) receptors blocked LTD. This effect was prevented by P2Y(1) receptor antagonists and was absent in mice lacking P2Y(1) but not P2Y(2) receptors. We also found that activation of P2Y(1) receptors inhibits Ca(2+) 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 P2Y(1) receptors. In conclusion, these data suggest that the reduction of Ca(2+) influx via VSCCs caused by the activation of P2Y(1) receptors by ATP is the possible mechanism for the inhibition of LTD in prefrontal cortex., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The expression and functionality of P2X/P2Y receptor subtypes in multipolar nonpyramidal neurons of mixed cortical cell cultures were investigated by means of immunocytochemistry and fura-2 microfluorimetry. The morphological studies revealed that most of the neurons are immunoreactive for GABA and express a range of P2X/P2Y receptors, predominantly of the P2X(2,4,6) and P2Y(1,2) subtypes. P2X(1) and P2X(7) receptor immunoreactivity (IR) was found on thin axon-like processes and presynaptic structures, respectively. Application of ATP caused a small concentration-dependent increase in intracellular Ca2+ concentration ([Ca2+]i) in most investigated neurons, whereas only about the half of these cells responded to 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP), ADPbetaS, 2MeSADP, or 2MeSATP and even fewer cells to UTP. In contrast, alpha,beta-meATP, UDP, and UDP-glucose failed to produce any [Ca2+]i signaling. The response to ATP itself was inhibited by pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), Reactive Blue 2, 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS2179), and suramin (300 microM) as well as by a cyclopiazonic acid-induced depletion of intracellular Ca2+ stores. A Ca2+-free external medium tended to decrease the ATP-induced [Ca2+]i transients, although this action did not reach statistical significance. Various blockers of voltage-sensitive Ca2+ channels and the gap junction inhibitor carbenoxolone did not interfere with the effect of ATP, whereas a combination of the ionotropic glutamate receptor antagonists D(-)-2-amino-5-phosphonopentanoic acid (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) decreased it. Cross-desensitization experiments between ADPbetaS or UTP and ATP suggested that ATP acts on the one hand via P2Y(1,2) receptors and on the other hand by additional signaling mechanisms. These mechanisms may involve the release of glutamate (which in consequence activates ionotropic glutamate receptors) and the entry of Ca2+ via store-operated Ca2+ channels. Evidence for the presence of functional P2X receptors, in particular P2X(7), remains elusive.

Nucleotides play an important role in brain development and may exert their action via ligand-gated cationic channels or G protein-coupled receptors. Patch-clamp measurements indicated that in contrast to AMPA, ATP did not induce membrane currents in human midbrain derived neuronal progenitor cells (hmNPCs). Various nucleotide agonists concentration-dependently increased [Ca(2+)](i) as measured by the Fura-2 method, with the rank order of potency ATP>ADP>UTP>UDP. A Ca(2+)-free external medium moderately decreased, whereas a depletion of the intracellular Ca(2+) storage sites by cyclopiazonic acid markedly depressed the [Ca(2+)](i) transients induced by either ATP or UTP. Further, the P2Y(1) receptor antagonistic PPADS and MRS 2179, as well as the nucleotide catalyzing enzyme apyrase, allmost abolished the effects of these two nucleotides. However, the P2Y(1,2,12) antagonistic suramin only slightly blocked the action of ATP, but strongly inhibited that of UTP. In agreement with this finding, UTP evoked the release of ATP from hmNPCs in a suramin-, but not PPADS-sensitive manner. Immunocytochemistry indicated the co-localization of P2Y(1,2,4)-immunoreactivities (IR) with nestin-IR at these cells. In conclusion, UTP may induce the release of ATP from hmNPCs via P2Y(2) receptor-activation and thereby causes [Ca(2+)](i) transients by stimulating a P2Y(1)-like receptor.

Mixed neuronal/astrocytic cortical cell cultures of the rat were incubated for 2 or 12h under normoxic or ischemic conditions. Subsequent flow cytometric analysis with an anti-P2X7 receptor antibody directed against an extracellular epitope indicated the up-regulation of these receptors at the plasma membrane by 12h of ischemia. Labelling of MAP-2 immunopositive neurons by an anti-P2X7 antibody directed against a C-terminal epitope, documented the selectivity of the ischemia-induced increase in receptor-density for the neuronal population. By contrast, staining of GFAP immunopositive astrocytes by the same anti-P2X7 antibody excluded any effect of ischemia on the astrocytic density of P2X7 receptors. The ischemic up-regulation of neuronal P2X7 receptors is in perfect agreement with the previously reported facilitation of transmitter release from the GABAergic non-pyramidal cell type in such cultures [K. Wirkner, A. Köfalvi, W. Fischer, A. Günther, H. Franke, H. Gröger-Arndt, W. Nörenberg, E. Madarasz, E.S. Vizi, D. Schneider, B. Sperlagh, P. Illes, Supersensitivity of P2X7 receptors in cerebrocortical cell cultures after in vitro ischemia, J. Neurochem. 95 (2005) 1421-1437].

Whole cell patch clamp investigations were carried out to clarify the pH sensitivity of native and recombinant P2X(3) receptors. In HEK293 cells permanently transfected with human (h) P2X(3) receptors (HEK293-hP2X(3) cells), an acidic pH shifted the concentration-response curve for alpha,beta-methylene ATP (alpha,beta-meATP) to the right and increased its maximum. An alkalic pH did not alter the effect of alpha,beta-meATP. Further, a low pH value increased the activation time constant (tau(on)) of the alpha,beta-meATP current; the fast and slow time constants of desensitization (tau(des1), tau(des2)) were at the same time also increased. Finally, acidification accelerated the recovery of P2X(3) receptors from the desensitized state. Replacement of histidine 206, but not histidine 45, by alanine abolished the pH-induced effects on hP2X(3) receptors transiently expressed in HEK293 cells. Changes in the intracellular pH had no effect on the amplitude or time course of the alpha,beta-meATP currents. The voltage sensitivity and reversal potential of the currents activated by alpha,beta-meATP were unaffected by extracellular acidification. Similar effects were observed in a subpopulation of rat dorsal root ganglion neurons expressing homomeric P2X(3) receptor channels. It is suggested that acidification may have a dual effect on P2X(3) channels, by decreasing the current amplitude at low agonist concentrations (because of a decrease in the rate of activation) and increasing it at high concentrations (because of a decrease in the rate of desensitization). Thereby, a differential regulation of pain sensation during e.g. inflammation may occur at the C fiber terminals of small DRG neurons in peripheral tissues.

Extracellular adenosine 5'-triphosphate (ATP) was proposed to be an activity-dependent signaling molecule that regulates glia-glia and glia-neuron communications. ATP is a neurotransmitter of its own right and, in addition, a cotransmitter of other classical transmitters such as glutamate or GABA. The effects of ATP are mediated by two receptor families belonging either to the P2X (ligand-gated cationic channels) or P2Y (G protein-coupled receptors) types. P2X receptors are responsible for rapid synaptic responses, whereas P2Y receptors mediate slow synaptic responses and other types of purinergic signaling involved in neuronal damage/regeneration. ATP may act at pre- and postsynaptic sites and therefore, it may participate in the phenomena of long-term potentiation and long-term depression of excitatory synaptic transmission. The release of ATP into the extracellular space, e.g., by exocytosis, membrane transporters, and connexin hemichannels, is a widespread physiological process. However, ATP may also leave cells through their plasma membrane damaged by inflammation, ischemia, and mechanical injury. Functional responses to the activation of multiple P2 receptors were found in neurons and glial cells under normal and pathophysiological conditions. P2 receptor-activation could either be a cause or a consequence of neuronal cell death/glial activation and may be related to detrimental and/or beneficial effects. The present review aims at demonstrating that purinergic mechanisms correlate with the etiopathology of brain insults, especially because of the massive extracellular release of ATP, adenosine, and other neurotransmitters after brain injury. We will focus in this review on the most important P2 receptor-mediated neurodegenerative and neuroprotective processes and their beneficial modulation by possible therapeutic manipulations.

P2X(7) 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 P2X(7) receptor, as well as its cell-type specific localization in the nervous system. Subsequently, the participation of P2X(7) 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 P2X(7) receptors are also discussed.

Extracellular adenosine 5'-triphosphate (ATP) has been recognized as a ubiquitous, unstable signalling molecule, acting as a fast neurotransmitter and modulator of transmitter release and neuronal excitability. Recent findings have demonstrated that ATP is a growth factor participating in differentiation, cell proliferation, and survival, as well as a toxic agent that mediates cellular degeneration and death. Potential sources of extracellular purines in the nervous system include neurons, glia, endothelium, and blood. A complex family of ectoenzymes rapidly hydrolyzes or interconverts extracellular nucleotides, thereby either terminating their signalling action or producing an active metabolite of altered purinoceptor selectivity. Most effects are mediated through the 2 main subclasses of specific cell surface receptors, P2X and P2Y. Members of these P2X/Y receptor families are widely expressed in the central nervous system (CNS) and are involved in glia-glia and glia-neuron communications, whereby they play important physiological and pathophysiological roles in a variety of biological processes. After different kinds of "acute" CNS injury (e.g., ischemia, hypoxia, mechanical stress, axotomy), extracellular ATP can reach high concentrations, up to the millimolar range, flowing out from cells into the extracellular space, exocytotically, via transmembrane transport, or as a result of cell damage. In this review, P2 receptor activation as a cause or a consequence of neuronal cell activation or death and/or glial activation is described. The involvement of P2 receptors is also described under different "chronic" pathological conditions, such as pain, epilepsia, toxic influence of ethanol or amphetamine, retinal diseases, Alzheimer's disease (AD), and possibly, Parkinson's disease. The relationship between changes in P2 receptor expression and the specific response of different cell types to injury is extremely complex and can be related to detrimental and/or beneficial effects. The present review therefore considers ATP acting via P2 receptors as a potent regulator of normal physiological and pathological processes in the brain, with a focus on pathophysiological implications of P2 receptor functions.

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.

Investigation of the role of extracellular nucleotides in nervous system has been one of the main topics of the P2Y receptor research throughout the years. In parallel to numerous studies on primary culture systems, various neuronal and non-neuronal cell lines have been used to model in vitro the processes mediated by extracellular nucleotides. In this review article, a survey of expression profiles of G protein-coupled P2Y receptor subtypes in nervous-system-derived cell lines is presented, by analysing the receptor expression at the mRNA, protein, and functional level. The variability of receptor expression profiles in established cell lines is further discussed, bringing forward some general properties for neuronal and glial malignant cell lines.

Whole-cell patch-clamp recordings from cultured rat dorsal root ganglion neurons demonstrated that the P2Y1 receptor agonists adenosine 5'-O-2-thiodiphosphate (ADP-beta-S) and 2-methylthio adenosine 5'-diphosphate (2-MeSADP) inhibit the alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-meATP)-induced P2X3 receptor-currents. This effect could be antagonized by the wide-spectrum G protein blocker GDP-beta-S and the P2Y(1) receptor antagonist MRS 2179. The P2Y12,13 receptor antagonist AR-C6993MX and pertussis toxin, a blocker of Galphai/o, did not interact with the effect of ADP-beta-S. Hence, the results indicate that ADP-sensitive P2Y1 receptors of rat dorsal root ganglion neurons inhibit ionotropic P2X3 receptors via G protein-activation.

1. The presence of ionotropic P2X receptors, targets of ATP in fast synaptic transmission, as well as metabotropic P2Y receptors, known to activate K(+) currents in cultured neostriatal neurones, was investigated in medium-spiny neurones and cholinergic interneurones contained in neostriatal brain slices from 5-26-day-old rats. 2. In these cells, adenosine-5'-triphosphate (ATP) (100-1000 microm), 2-methylthioadenosine-5'-triphosphate (2MeSATP), alpha,beta-methyleneadenosine-5'-triphosphate (alpha,betameATP, 30-300 microm, each) and adenosine-5'-O-(3-thiotriphosphate (ATPgammaS) (100 microm) failed to evoke P2X receptor currents even when 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microm), apyrase (10 U ml(-1)) or intracellular Cs(+) was used to prevent occluding effects of the ATP breakdown product adenosine, desensitisation of P2X receptors by endogenous ATP and an interference with the activation of K(+) channels, respectively. P2X receptor agonists were also ineffective in outside-out patches withdrawn from the brain slice tissue. Muscimol (10 microm) evoked GABA(A) receptor-mediated currents under all these conditions. 3. When used as a control, locus coeruleus neurones responded with P2X receptor-mediated currents to ATP (300 microm), 2MeSATP and alpha,betameATP (100 microm, each). 4. ATP and adenosine-5'-diphosphate (ADP) (100 microm, each) did not activate K(+) currents in the neostriatal neurones. 5. Despite the observed lack of function, P2X(2) and P2Y(1) immunofluorescence was found in roughly 50% of the medium-spiny neurones and cholinergic interneurones. 6. A role of ATP in synaptic transmission to striatal medium-spiny neurones and cholinergic interneurones appears unlikely, however, the otherwise silent P2X and P2Y receptors may gain functionality under certain yet unknown conditions.

The function of adenosine A(2A) receptors, localized at the enkephalin-containing GABAergic medium spiny neurons of the striatum, has been discussed controversially. Here we show that, in the absence of external Mg(2+), the adenosine A(2A) receptor agonist CGS 21680 postsynaptically depressed the NMDA, but not the non-NMDA (AMPA/kainate) receptor-mediated fraction of the electrically evoked EPSCs in a subpopulation of striatal neurons. Current responses to locally applied NMDA but not AMPA were also inhibited by CGS 21680. However, in the presence of external Mg(2+), the inhibition by CGS 21680 of the GABA(A) receptor-mediated IPSCs led to a depression of the EPSC/IPSC complexes. The current response to the locally applied GABA(A) receptor agonist muscimol was unaltered by CGS 21680. Whereas, the frequency of spontaneous (s)IPSCs was inhibited by CGS 21680, their amplitude was not changed. Hence, it is suggested that under these conditions the release rather than the postsynaptic effect of GABA was affected by CGS 21680. In conclusion, under Mg(2+)-free conditions, CGS 21680 appeared to postsynaptically inhibit the NMDA receptor-mediated component of the EPSC, while in the presence of external Mg(2+) this effect turned into a presynaptic inhibition of the GABA(A) receptor-mediated IPSC.

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, Ca2+ currents (I(Ca)). The rank order of agonist potency was UTP > ADP > ATP. ATP depressed the omega-conotoxin GVIA-sensitive N-type current only. Pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate tetraammonium, two P2Y1 receptor antagonists, almost abolished the ATP-induced inhibition. Both patch-clamp recordings and immunocytochemistry coupled with confocal laser microscopy indicated a colocalization of functional P2X3 and P2Y1 receptors on the same DRG neurons. Because the effect of ATP was inhibited by intracellular guanosine 5'-O-(2-thiodiphosphate) or by applying a strongly depolarizing prepulse, P2Y1 receptors appear to block I(Ca) by a pathway involving the betagamma subunit of a G(q/11) protein. Less efficient buffering of the intracellular Ca2+ concentration ([Ca2+]i) by reducing the intrapipette EGTA failed to interfere with the ATP effect. Fura-2 microfluorimetry suggested that ATP raised [Ca2+]i by a Galpha-mediated release from intracellular pools and simultaneously depressed the high external potassium concentration-induced increase of [Ca2+]i by inhibiting I(Ca) via Gbetagamma. Adenosine 5'-O-(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 effect of ATP.

Neurons of the central nervous system (CNS) are endowed with ATP-sensitive receptors belonging to the P2X (multimeric ligand-gated cationic channels) and P2Y (G protein-coupled 7TM receptors) types. To date seven P2X and eight 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. P2X(7)subunits do not form heterooligomeric assemblies and are uniqe in mediating apoptosis and necrosis of glial cells and possibly also of neurons. The P2X(2), P2X(4), P2X(4)/P2X(6) and P2Y(1) receptors appear to be the predominant neuronal 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. The localisation of these receptors may be at the terminal axons (presynaptic) or at the somato-dendritic region (postsynaptic). Whereas presynaptic P2 receptors may be either excitatory (P2X) or inhibitory (P2Y), postsynaptic P2 receptors appear to be without exception excitatory. Finally, the enzymatic degradation of ATP may lead to the local generation of adenosine which can modulate ATP-related neurotransmission via activation of A(1) or A(2A) receptors.

Aims and Methods: Immunocytochemical studies revealed that the vast majority of neurons in our primary cultures of rat cortical cells are GABA-positive and represent nonpyramidal interneuron-like cells. The influence of ethanol on (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)-induced Ca2+ influx was investigated in multipolar, medium-sized neurons by using single-cell fura-2 microfluorimetry., Results: In a first series of experiments, the results showed a small but significant decrease of 17-22% by ethanol (100 mm) of the intracellular Ca2+ signals induced by slowly superfused AMPA (10, 30, 100 microm). This finding is comparable with the inhibitory activity of ethanol on N-methyl-D-aspartic acid (NMDA)-induced Ca2+ signals in these cells. Further studies with a fast pressure-application of AMPA (30 microm) showed a similar degree of inhibition by ethanol (100 mm). Superfusion with tetrodotoxin/bicuculline, to rule out possible effects of spontaneously released GABA and synaptic spike activity, did not significantly influence the AMPA-induced Ca2+ response nor the inhibitory effect of ethanol., Conclusions: The present findings indicate that ethanol at high concentrations inhibits Ca2+ signaling via both AMPA and NMDA glutamate receptors in cortical interneuron-like cells. These effects may contribute to the central depressant action of this drug.

Mesencephalic dopaminergic neurons are known to project to the prefrontal cortex (PFC) and the striatum (STR). Organotypic slice co-cultures of the ventral tegmental area/substantia nigra (VTA/SN)-complex and the PFC or STR, respectively, were used to analyze the cytoarchitectural organization of the VTA/SN-complex and the innervation pattern of the target slices by dopaminergic fibers. After 10-28 days of culturing immunocytochemistry with antibodies against tyrosine hydroxylase (TH) was performed. The VTA/SN-complex revealed in vitro an organization of TH-positive cells similar to those observed in rat brains of comparable age. TH-immunoreactive cells exhibited their typical morphology and formed long processes. No TH-immunolabeled elements were found in single cultures of PFC and STR. Tracing of VTA/SN fibers with biocytin as well as TH-immunostaining showed numerous labeled fibers in the co-cultured slices. Extensive fiber crossing was observed in the co-cultures of the VTA/SN-complex and STR but only a sparse fiber bridge in the co-cultured slices of VTA/SN-complex and PFC. The VTA/SN-complex-PFC system obviously retained several of its in vivo characteristics, e.g. the fiber network in the prefrontal cortical subareas. Our results demonstrate that TH-immunoreactive neurons develop their typical innervation pattern in slice co-cultures of VTA/SN-complex and PFC or STR, respectively. This in vitro approach may be useful for investigations of the dopaminergic function in the VTA/SN-prefrontal pathway.

The aim of the present study was to investigate whether 5,7-dihydroxytryptamine (5,7-DHT), an autofluorescent serotonin derivative, can be used as a specific marker for serotonergic or dopaminergic neurons in rat mesencephalic cultures. To this end, primary cultures were prepared from the ventral brain stem of 14-day-old Wistar rat foetuses and kept in culture for 10 days (DIV10). At DIV10, the cultures were characterized immunocytochemically with antibodies raised against tyrosine hydroxylase (TH; a marker for catecholaminergic/ dopaminergic neurons) and serotonin (5-HT). 5,7-DHT labelling of the neurons was investigated after incubation with 25 microM of the serotonin derivative (plus 0.005% ascorbic acid) for 60 min at 37 degrees C, followed by incubation with primary antibodies against TH or serotonin and a fluorescence (Cy3)-labelled secondary antibody. Using confocal laser scanning microscopy, this double immunofluorescence approach demonstrated that all cells which had accumulated 5,7-DHT additionally displayed anti-5-HT immunoreactivity, whereas no evidence was found for 5,7-DHT labelling of TH immunoreactive cells. Preincubation with the selective serotonin reuptake inhibitor fluvoxamine maleate (10 microM) prevented the loading of the 5-HT-positive cells with 5,7-DHT. In conclusion, the present data indicate that 5,7-DHT specifically labels serotonergic cells in rat midbrain cultures. Thus, 5,7-DHT can be used for the identification of living serotonergic neurons even in the presence of dopaminergic neurons.

The effects of the adenosine A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine (CGS 21680) on currents mediated by excitatory amino acid receptors were examined in rat striatal brain slices. In a Mg(2+)-free superfusion medium, CGS 21680 decreased the amplitude of excitatory postsynaptic currents (EPSCs) in about 70% of striatal neurons. The inhibitory effect of CGS 21680 disappeared both in the presence of the adenosine A(2A) receptor antagonist 8-(3-chlorostyryl) caffeine and the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP-5). NMDA-induced currents were also depressed by CGS 21680 in a subset of striatal cells, whereas alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-induced currents were not affected. The results suggest that adenosine A(2A) receptor agonists inhibit the NMDA component of the EPSC.

Vanilloid receptors (VR) integrate various painful stimuli, e.g., noxious heat, acidic pH, capsaicin, and resiniferatoxin (RTX). Although VR antagonists may be useful analgesics, the available agents capsazepine and ruthenium red lack the necessary potency and selectivity. Recently, submicromolar concentrations of the arginine-rich hexapeptide RRRRWW-NH(2) (R(4)W(2)) blocked VR-mediated ionic currents in a Xenopus expression system in a noncompetitive and nonstereoselective manner. Here, VR-antagonistic effects of L-R(4)W(2) and D-R(4)W(2), hexapeptides consisting entirely of L- and D-amino acids, were characterized in native adult rat dorsal root ganglion neurons using [Ca(2+)](i) imaging (Fura-2/acetoxymethyl ester). Fura-2 fluorescence ratio (R) was increased by RTX and capsaicin by 0.473 +/- 0.098 unit above basal levels of 0.903 +/- 0.011 (R(max), 2.289 +/- 0.031; R(min), 0.657 +/- 0.007) in a concentration-dependent manner (log EC(50): RTX, -10.04 +/- 0.05, n = 10; capsaicin, -6.60 +/- 0.10, n = 11). Agonist concentration-response curves were shifted to the right by L- and D-R(4)W(2) (0.1, 1, and 10 microM each) and by capsazepine (3, 10, 30, and 100 microM), whereas their maximal effects and slopes remained unaffected, indicating competitive antagonism. Schild analysis for L-R(4)W(2) yielded apparent dissociation constants of 4.0 nM (RTX) and 3.7 nM (capsaicin), and slopes smaller than unity (RTX, 0.38; capsaicin, 0.42). Apparent dissociation constants and slopes for D-R(4)W(2) and capsaicin were 153 nM and 0.67 versus 4.1 microM and 1.19 for capsazepine and capsaicin. Thus, VR-mediated effects in native dorsal root ganglion neurons were antagonized by L-R(4)W(2) > D-R(4)W(2) > capsazepine (order of potency). In conclusion, the R(4)W(2) hexapeptide is a potent, stereospecific, and (probably) competitive VR antagonist, although an allosteric interaction cannot be completely ruled out.

Immunocytochemical and Co(2+) uptake studies revealed that in primary cultures of rat cortical neurones, the majority of neurones are gamma-aminobutyric acid (GABA) immunopositive and can express Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors. By fura-2 microfluorimetry, it was shown that the stimulation with the selective agonist (S)-AMPA (0.3-300 microM) induced a concentration-dependent but cell-variable increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) (EC(50) value 7.4 microM) in more than 80% of the medium-sized multipolar neurones studied. The AMPA-induced rise in [Ca(2+)](i) seems to be due to Ca(2+) entry through AMPA receptor channels, because the response was abolished in Ca(2+)-free solution and by AMPA receptor selective antagonists, but was not significantly influenced by cyclopiazonic acid, an inhibitor of the endoplasmatic Ca(2+)-ATPase, by selective N-methyl-D-aspartic acid (NMDA) receptor antagonists, as well as the Na(+) channel blocker tetrodotoxin and the majority of tested Ca(2+) channel blockers. In conclusion, the results indicate that the cerebral cortical neurones in culture represent mostly GABAergic interneurone-like cells and the majority of them possess Ca(2+)-permeable AMPA receptors, important for intracellular signal transduction and neuronal plasticity.

Mixed striatal cell cultures containing neurons and glial cells were grown either in neurobasal medium (NBM) or Dulbecco's modified Eagle's medium (DMEM). Whole-cell patch-clamp recordings indicated that, if at all, only a single, low amplitude spike was evoked shortly after starting the injection of a depolarizing current pulse into NBM neurons. In contrast, DMEM neurons fired series of high amplitude action potentials, without apparent spike frequency adaptation. The possible reason for the observed action potential failure in NBM neurons was a low density of Na+ channels per unit of membrane surface area. However, both in NBM and DMEM neurons, ATP did not induce inward current responses via P2X receptor-channels, although GABAA and N-methyl-D-aspartate (NMDA) receptor-channels could be activated by muscimol and NMDA, respectively. Ca2+ imaging experiments by means of the Fura-2 method were utilized to measure intracellular Ca2+ ([Ca2+]i) in neurons and glial cells. NBM, but not DMEM neurons responded to ATP with [Ca2+]i transients; glial cells grown in either culture medium were equally sensitive to ATP. ATP caused an increase of [Ca2+]i by a mechanism only partly dependent on external Ca2+; the residual ATP effect was blocked by cyclopiazonic acid (CPA) and was therefore due to the release of Ca2+ from its intracellular pools. The receptor involved was characterized by P2 receptor antagonists (PPADS, MRS 2179, AR-C69931MX) and was found to belong to the P2Y1 subtype. CPA caused an early [Ca2+]i response due to release from intracellular storage sites, followed by a late [Ca2+]i response due to the influx of this cation from the extracellular space, probably triggered by the opening of store-operated channels (SOCs) in the plasma membrane. It is concluded that in partial analogy with the effect of CPA, ATP releases [Ca2+]i via the Gq/phospholipase C/inositoltrisphosphate (IP3) pathway, thereby opening SOCs. It is hypothesized that this effect of ATP may have an important role for the proliferation and migration of striatal neuronal progenitors. J. Cell. Physiol. 209: 81–93, 2006. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

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]

1 Vasoactive intestinal peptide (VIP) is present in the hippocampus in three subtypes of GABAergic interneurones, two of which innervate preferentially other interneurones, responsible for pyramidal cell inhibition. We investigated how pre- and postsynaptic modulation of GABAergic transmission (to both pyramidal cells and interneurones) by VIP could influence excitatory synaptic transmission in the CA1 area of the hippocampus. 2 VIP (0.1-100 nM) increased [³H]GABA release from hippocampal synaptosomes (maximum effect at 1 nM VIP; 6.3.8 ± 4.0%) but did not change [³H]glulamate release. 3 VIP (0.3-30nM) enhanced synaptic transmission in hippocampal slices (maximum effect at 1 nM VIP; field excitatory postsynaptic potentials (epsp) slope: 23 7 ± 1.1%; population spike amplitude: 20.3 ± 1.7%). The action on field epsp slope was fully dependent on GABAergic transmission since it was absent in the presence of picrotoxin (50 μM) plus CGP55845 (1 μM). 4 VIP (1 nM) did not change paired-pulse facilitation but increased paired-pulse inhibition in CA1 pyramidal cells (16.0 ± 0.9%), reinforcing the involvement of GABAergic transmission in the action of VIP. 5 VIP (1 nM) increased muscimol-evoked inhibitory currents by 36.4 ± 8.7% in eight out often CA1 interneurones in the siraiuin radianini. This suggests that VIP promotes increased inhibition of interneurones that control pyramidal cells, leading to disinhibition of synapiic transmission to pyramidal cell dendrites. 6 In conclusion, concerted pre- and postsynaptic actions of VIP lead to disinhibition of pyramidal cell dendrites causing an enhancement of synaptic transmission. [ABSTRACT FROM AUTHOR]

1. The presence of ionotropic P2X receptors, targets of ATP in fast synaptic transmission, as well as metabotropic P2Y receptors, known to activate K+ currents in cultured neostriatal neurons, was invistifated in medium-spiny neutrons and cholinergic interneurones contained in neostriatal brain slices from 5-26-day-old rats. 2. In these cells, adenosine-5'-triphosphate (ATP) (100-1000 µM), 2-methylthioadenosine-5'-triphosphate (2MeSATP), α,β-methuleneadenosine-5'-triphosphate (α,βmeATP, 30-300 µM, each) and adenosine-5'-O-(3-thiotriphosphate (ATPγS) (100 µM) failed to evoke P2X receptor currents even when 8-cyclopentyl-1,3,-dipropylxanthine (DPCPX, 0.1 µM), apyrase (10 U ml-1) or intracellular Cs+ was used to prevent occluding effects of the ATP breakdown product adenosine, desensitization of P2X receptors by endogenous ATP and an interference with the activation of K+ channels, respectively. P2X receptor agonists were also ineffective in outside-out patches withdrawn from the brain slice tissue. Muscimol (10 µM) evoked GABAA receptor-mediated currents under all these conditions. 3. When used as a control, locus coeruleus neurons responded with P2X receptor-mediated currents for ATP (300 µM) each. 4. ATP and adenosine-5'-diphosphate (ADP) (100 µM, each) did not activate K+ currents in the neostriatal neutrons. 5. Despite the observed lack of function, P2X2 and P2Y1 immunofluoroscence was found in roughly 50% of the medium-spiny neurons and cholinergic interneutrones. 6. A role of ATP in synaptic transmission to striatal medium-spiny neutrons and cholinergic interneurones appears unlikely, however, the otherwise silent P2X and P2Y receptors may gain functionality under-certain yet unknown conditions. [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]

Whole-cell patch-clamp recordings were performed on 12- to 15-day-old rat locus coeruleus neurones in a midpontine slice preparation. Application of noradrenaline (100 µM) and N-methyl-D-aspartate (NMDA; 100 µM) induced a small outward current and a distinct inward current, respectively. Single-cell reverse transcriptase-polymerase chain reaction (scRT-PCR), used to analyse the expression pattern of NMDA receptor subunits 2A, 2B, and 2C (NR2A–C) subsequent to electrophysiological characterization, demonstrated differences in the capacity of individual locus coeruleus neurones to express NR2A–C mRNA. NR2C mRNA expression predominated over those of NR2A and NR2B mRNA in most neurones. In addition, in neurones containing NR2C mRNA NMDA induced significantly larger currents than in cells lacking expression of this gene. RT-PCR studies performed on tissue preparations of adult rats also revealed a distinct expression of NR2C mRNA. In conclusion, the present data demonstrate differences in the mRNA expression pattern of NR2A–C of individual locus coeruleus neurones with a predominant NR2C mRNA expression in the majority of the cells. [ABSTRACT FROM AUTHOR]

Abstract: Whole-cell patch-clamp recordings from cultured rat dorsal root ganglion neurons demonstrated that the P2Y1 receptor agonists adenosine 5′-O-2-thiodiphosphate (ADP-β-S) and 2-methylthio adenosine 5′-diphosphate (2-MeSADP) inhibit the α,β-methylene adenosine 5′-triphosphate (α,β-meATP)-induced P2X3 receptor-currents. This effect could be antagonized by the wide-spectrum G protein blocker GDP-β-S and the P2Y1 receptor antagonist MRS 2179. The P2Y12,13 receptor antagonist AR-C6993MX and pertussis toxin, a blocker of Gαi/o, did not interact with the effect of ADP-β-S. Hence, the results indicate that ADP-sensitive P2Y1 receptors of rat dorsal root ganglion neurons inhibit ionotropic P2X3 receptors via G protein-activation. [Copyright &y& Elsevier]

The whole-cell patch-clamp technique was used to record current responses to AMPA, N-methyl- d -aspartate (NMDA), muscimol and dibenzoyl-ATP (Bz-ATP) in superficial (reactive/gliotic) substantia gelatinosa (SG) astrocytes and neurons of spinal cord slices kept for different periods of time in organotypic culture. Currents induced by AMPA, NMDA and muscimol confirmed the existence of their specific receptors in 2-week-old neurons; astrocytes cultured for the same period of time responded to AMPA and muscimol, but not to NMDA. AMPA had a larger effect on 2-week-old astrocytes than on the 1-week-old ones, in spite of a similar sensitivity of the age-matched neurons to this amino acid. The effect of the prototypic P2X7 receptor agonist Bz-ATP on superficial astrocytes and neurons depended on the drug concentration applied and increased in parallel with the lengthening of the culture period. The amplitudes of Bz-ATP currents of deep (resting) astrocytes were age-independent. Neurons located in deep layers exhibited after 1 week of culturing much larger Bz-ATP currents than the superficial ones of the same age. In conclusion, whereas resting astrocytes had culture period-independent P2X7 receptor-sensitivity, reactive/gliotic astrocytes exhibited P2X7 receptor-sensitivity increasing in parallel with the prolongation of the time spent in culture. The results with Bz-ATP agree with the facilitation of AMPA-induced currents in reactive astrocytes during development, and with the hypothesis that extracellular ATP is an ontogenetically early transmitter/signaling molecule in the CNS. [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: Membrane currents of layer V pyramidal cells in slices of the rat prefrontal cortex (PFC) were recorded with the patch-clamp technique. In an Mg2+-free superfusion medium l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), a preferential blocker of astrocytic glutamate transporters, caused inward current due to the activation of NMDA receptors. The blockade of conducted action potentials by tetrodotoxin did not interfere with this effect. ATP was inactive when given alone and potentiated the NMDA-induced current in an Mg2+-containing but not Mg2+-free superfusion medium. Agonists of group I ((S)-3,5-dihydroxyphenylglycine; DHPG) and II ((1R,4R,5S,6R)-4-amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid; LY 379268) metabotropic glutamate receptors (mGluRs) also potentiated responses to NMDA, whereas the group III mGluR agonist l-(+)-2-amino-4-phosphonobutyric acid (l-AP4) did not affect them. In contrast to ATP, PDC evoked inward current in the absence but not in the presence of external Mg2+, when given alone, and facilitated the NMDA effect Mg2+-independently. The PDC-induced facilitation of NMDA responses was blocked by group II ((2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid; LY 341495), but not group I ((RS)-1-aminoindan-1,5-dicarboxylic acid; AIDA) or III (α-methyl-3-methyl-4-phosphonophenylglycine; UBP 1112) mGluR antagonists. In conclusion, the blockade of astrocytic glutamate uptake by PDC may lead to a stimulation of group II mGluRs, while the triggering of exocytotic glutamate release from astrocytes by ATP may cause activation of group I mGluRs, both situated postsynaptically at layer V PFC pyramidal cells. Either group of mGluRs may interact with NMDA receptors in a positive manner. [Copyright &y& Elsevier]

Whole cell patch clamp investigations were carried out to clarify the pH sensitivity of native and recombinant P2X3 receptors. In HEK293 cells permanently transfected with human (h) P2X3 receptors (HEK293-hP2X3 cells), an acidic pH shifted the concentration-response curve for α,β-methylene ATP (α,β-meATP) to the right and increased its maximum. An alkalic pH did not alter the effect of α,β-meATP. Further, a low pH value increased the activation time constant (̄on) of the α,β-meATP current; the fast and slow time constants of desensitization (̄des1, ̄des2) were at the same time also increased. Finally, acidification accelerated the recovery of P2X3 receptors from the desensitized state. Replacement of histidine 206, but not histidine 45, by alanine abolished the pH-induced effects on hP2X3 receptors transiently expressed in HEK293 cells. Changes in the intracellular pH had no effect on the amplitude or time course of the α,β-meATP currents. The voltage sensitivity and reversal potential of the currents activated by α,β-meATP were unaffected by extracellular acidification. Similar effects were observed in a subpopulation of rat dorsal root ganglion neurons expressing homomeric P2X3 receptor channels. It is suggested that acidification may have a dual effect on P2X3 channels, by decreasing the current amplitude at low agonist concentrations (because of a decrease in the rate of activation) and increasing it at high concentrations (because of a decrease in the rate of desensitization). Thereby, a differential regulation of pain sensation during e.g. inflammation may occur at the C fiber terminals of small DRG neurons in peripheral tissues. [ABSTRACT FROM AUTHOR]

The function of adenosine A2A receptors, localized at the enkephalin-containing GABAergic medium spiny neurons of the striatum, has been discussed controversially. Here we show that, in the absence of external Mg2+, the adenosine A2A receptor agonist CGS 21680 postsynaptically depressed the NMDA, but not the non-NMDA (AMPA/kainate) receptor-mediated fraction of the electrically evoked EPSCs in a subpopulation of striatal neurons. Current responses to locally applied NMDA but not AMPA were also inhibited by CGS 21680. However, in the presence of external Mg2+, the inhibition by CGS 21680 of the GABAA receptor-mediated IPSCs led to a depression of the EPSC/IPSC complexes. The current response to the locally applied GABAA receptor agonist muscimol was unaltered by CGS 21680. Whereas, the frequency of spontaneous (s)IPSCs was inhibited by CGS 21680, their amplitude was not changed. Hence, it is suggested that under these conditions the release rather than the postsynaptic effect of GABA was affected by CGS 21680. In conclusion, under Mg2+-free conditions, CGS 21680 appeared to postsynaptically inhibit the NMDA receptor-mediated component of the EPSC, while in the presence of external Mg2+ this effect turned into a presynaptic inhibition of the GABAA receptor-mediated IPSC. [Copyright &y& Elsevier]

The application of cyclophosphamide to rats was used to induce interstitial cystitis. Behavioural studies indicated a strong pain reaction that developed within 2 h and levelled off thereafter causing a constant pain during the following 18 h. Neurons prepared from L6/S1 dorsal root ganglia innervating the urinary bladder responded to the application of capsaicin or α,β-methylene ATP (α,β-meATP) with an increase of intracellular Ca2+ ([Ca2+]i). The [Ca2+]i responses to capsaicin were identical in the dorsal root ganglion cells of cyclophosphamide- and saline-treated rats, whereas α,β-meATP induced less increase in [Ca2+]i in the cyclophosphamide-treated animals than in their saline-treated counterparts. Hence, α,β-meATP-sensitive P2X3 and/or P2X2/3 receptors of L6/S1 dorsal root ganglion neurons were functionally downregulated during subacute pain caused by experimental cystitis. In contrast, capsaicin-sensitive vanilloid 1 receptors did not react to the same procedure. Thoracal dorsal root ganglia, not innervating the urinary bladder, were also unaltered in their responsiveness to α,β-meATP by cyclophosphamide treatment. [Copyright &y& Elsevier]