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Although previous studies described the production of IgG antibodies in a subgroup of patients with common variable immunodeficiency (CVID) following messenger RNA vaccinations with BNT162b2 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (CVID responders), the functionality of these antibodies in terms of avidity as measured by the dissociation rate constant (k dis) and the antibody response to booster immunization has not been studied. We sought to analyze in CVID responders and healthy individuals, the avidity of anti–SARS-CoV-2 serum antibodies and their neutralization capacity as measured by surrogate virus–neutralizing antibodies in addition to IgG-, IgM-, and IgA-antibody levels and the response of circulating (peripheral blood) follicular T-helper cells after a third vaccination with BNT162b2 SARS-CoV-2 messenger RNA vaccine. Binding IgG, IgA, and IgM serum levels were analyzed by ELISA in patients with CVID responding to the primary vaccination (CVID responders, n = 10) and healthy controls (n = 41). The binding avidity of anti–spike antibodies was investigated using biolayer interferometry in combination with biotin-labeled receptor-binding-domain of SARS-CoV-2 spike protein and streptavidin-labeled sensors. Antigen-specific recall T-cell responses were assessed by measuring activation-induced markers by flow cytometry. After the third vaccination with BNT162b2, IgG-, IgM-, and IgA-antibody levels, surrogate virus–neutralizing antibody levels, and antibody avidity were lower in CVID responders than in healthy controls. In contrast, anti–SARS-CoV-2 spike protein avidity was comparable in CVID responders and healthy individuals following primary vaccination. Follicular T-helper cell response to booster vaccination in CVID responders was significantly reduced when compared with that in healthy individuals. Impaired affinity maturation during booster response provides new insight into CVID pathophysiology. [ABSTRACT FROM AUTHOR]

Human gut is in permanent contact with microorganisms that play an important role in many physiological processes including metabolism and immunologic activity. These microorganisms communicate and manage themself by the quorum sensing system (QS) that helps to coordinate optimal growth and subsistence by activating signaling pathways that regulate bacterial gene expression. Diverse QS molecules produced by pathogenic as well as resident microbiota have been found throughout the human gut. However, even a host can by affected by these molecules. Intestinal and immune cells possess a range of molecular targets for QS. Our present knowledge on bacteria-cell communication encompasses G-protein-coupled receptors, nuclear receptors and receptors for bacterial cell-wall components. The QS of commensal bacteria has been approved as a protective factor with favourable effects on intestinal homeostasis and immunity. Signaling molecules of QS interacting with above-mentioned receptors thus parcipitate on maintaining of barrier functions, control of inflammation processes and increase of resistance to pathogen colonization in host organisms. Pathogens QS molecules can have a dual function. Host cells are able to detect the ongoing infection by monitoring the presence and changes in concentrations of QS molecules. Such information can help to set the most effective immune defence to prevent or overcome the infection. Contrary, pathogens QS signals can target the host receptors to deceive the immune system to get the best conditions for growth. However, our knowledge about communication mediated by QS is still limited and detailed understanding of molecular mechanisms of QS signaling is desired. • QS molecules affect the human organism through interactions with cellular receptors. • QS signals of commensal microbiota are beneficial for gut homeostasis and immunity. • Recognizing pathogen's QS molecules by host leads to the effective immune response. • Pathogen's QS signals can trick the host immune system to promote infection. [ABSTRACT FROM AUTHOR]

The function of almost all cells of the human and animal body is synchronized by purinergic/pyrimidinergic extracellular signalling molecules. This network activity is especially efficient in the central and peripheral nervous systems, driven by secretion of the (co)transmitter ATP (including its enzymatic degradation products ADP, AMP, and adenosine), as well as ATP/UTP (including UDP) released from the cytoplasm by either Ca2+-dependent vesicular exocytosis or by non-exocytotic pathways via a family of diverse channels. It must be pointed out that neural cells (neurons, astrocytes, and oligodendrocytes) are equal sources of nucleotides/nucleosides, as non-neural cells (e.g. the endothelium of small blood vessels). A whole plethora of purinergic receptors responding to the endogenously released purine and pyrimidine nucleotides as well as to adenosine, are instrumental in providing the structural basis for cell stimulation. The present collection of papers summarizes current knowledge and recent findings in the medicinal chemistry, electrophysiology, neuropharmacology and neurobiology of purinergic transmission. Accruing evidence supports the key role of extracellular nucleotides and nucleosides in neuroinflammation, neurodegeneration, and in neuropsychiatric diseases, thus paving the way for pharmacological intervention thanks to the development of novel brain-permeant, drug-like, purinergic ligands. We are confident that these therapies will open a new avenue for the treatment of so far uncurable diseases of the central and peripheral nervous systems. [ABSTRACT FROM AUTHOR]

Background: Implantable Cardiac Monitors (ICMs) are used for long-term monitoring of arrhythmias. BIOMONITOR III is a novel ICM with a miniaturized profile, long sensing vector due to a flexible antenna, simplified implantation with a dedicated insertion tool for pocket formation and ICM placement in a single step, and daily automatic Home Monitoring (HM) function.Methods: In 47 patients undergoing BIOMONITOR III insertion for any ICM indication, 16 investigators at 10 Australian sites assessed handling characteristics of the insertion tool, R-wave amplitudes, noise burden, P-wave visibility, and HM transmission success. Patients were followed for 1 month.Results: All 47 attempted insertions were successful. Median time from skin incision to removal of the insertion tool after ICM insertion was 39 s (IQR 19-65) and to wound closure and cleaning was 4.7 min (IQR 3.5-7.8). All aspects of the insertion tool were rated as "good" or "excellent" in ≥97.9% and "fair" in ≤2.1% of patients, except for "force needed for tunnelling" (91.5% good/excellent, 8.5% fair). Based on HM data, R-waves in the first month were stable at 0.70 ± 0.37 mV. Median noise burden (disabling automatic rhythm evaluation) was 0.19% (IQR 0.00-0.93), equivalent to 2.7 min (IQR 0.0-13.4) per day. In HM-transmitted ECG strips with regular sinus rhythm, P-waves were visible in 89 ± 24% of heart cycles. Patient-individual automatic Home Monitoring transmission success was 98.0% ± 5.5%.Conclusions: The novel ICM performed well in all aspects studied, including fast insertion, reliable R-wave sensing, good P-wave visibility, and highly successful HM transmissions. [ABSTRACT FROM AUTHOR]

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. In contrast to the peripheral nervous system, the functional significance of ATP in exerting rapid, excitatory (co)transmitter effects in the CNS appears to be relatively limited. ATP may be released from glial cells by exocytotic and nonexocytotic mechanisms. In the CNS, astrocytes may be interposed between neuronal afferents/interneurons and effector neurons to exert slow modulation of neuronal circuits by releasing the gliotransmitters glutamate, GABA, ATP, D-serine, and taurine. Gliotransmitter ATP is released from astrocytes in conjunction with several additional signaling molecules (cytokines, chemokines, free radicals, etc.) to modulate neuronal circuits. Although not discussed in the present review, neuroglial cells other than astrocytes (oligodendrocytes, Müller cells, Bergmann glia, etc.) as well as microglia also release ATP, and thereby exert effects similar to those of their astrocytic counterparts. [ABSTRACT FROM AUTHOR]

Purinergic signalling, consisting of extracellular purines and purinergic receptors, modulates cell proliferation, invasion and immunological reaction during cancer progression. Here, we focus on current evidence that suggests the crucial role of purinergic signalling in mediating cancer therapeutic resistance, the major obstacle in cancer treatment. Mechanistically, purinergic signalling can modulate the tumor microenvironment (TME), epithelial-mesenchymal transition (EMT) and anti-tumor immunity, thus affecting drug sensitivity of tumor cells. Currently, some agents attempting to target purinergic signalling either in tumor cells or in tumor-associated immune cells are under preclinical or clinical investigation. Moreover, nano-based delivery technologies significantly improve the efficacy of agents targeting purinergic signalling. In this review article, we summarize the mechanisms of purinergic signalling in promoting cancer therapeutic resistance and discuss the potentials and challenges of targeting purinergic signalling in future cancer treatment. [ABSTRACT FROM AUTHOR]

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’. [ABSTRACT FROM AUTHOR]

ATP (adenosine 5′-triphosphate), one of the most ancient neurotransmitters, exerts essential functions in the brain, including neurotransmission and modulation of synaptic activity. Moreover, this nucleotide has been attributed with trophic properties and experimental evidence points to the participation of ATP-activated P2X and P2Y purinergic receptors in embryonic brain development as well as in adult neurogenesis for maintenance of normal brain functions and neuroregeneration upon brain injury. We discuss here the available data on purinergic P2 receptor expression and function during brain development and in the neurogenic zones of the adult brain, as well as the insights based on the use of in vitro stem cell cultures. While several P2 receptor subtypes were shown to be expressed during in vitro and in vivo neurogenesis, specific functions have been proposed for P2Y1, P2Y2 metabotropic as well as P2X2 ionotropic receptors to promote neurogenesis. Further, the P2X7 receptor is suggested to function in the maintenance of pools of neural stem and progenitor cells through induction of proliferation or cell death, depending on the microenvironment. Pathophysiological actions have been proposed for this receptor in worsening damage in brain disease. The P2X7 receptor and possibly additional P2 receptor subtypes have been implicated in pathophysiology of neurological diseases including Parkinson's disease, Alzheimer's disease and epilepsy. New strategies in cell therapy could involve modulation of purinergic signaling, either in the achievement of more effective protocols to obtain viable and homogeneous cell populations or in the process of functional engraftment of transplanted cells into the damaged brain. This article is part of the Special Issue entitled ‘Purines in Neurodegeneration and Neuroregeneration’. [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]

Highlights: [•] ATP is released by various mechanisms following acute and chronic damage to the central nervous system. [•] Extracellular ATP activates P2X and P2Y receptors at astrocytes. [•] Activation of astrocytic P2X and P2Y receptors induced astrogliosis. [Copyright &y& Elsevier]

Abstract: 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 [Ca2+]i as measured by the Fura-2 method, with the rank order of potency ATP>ADP>UTP>UDP. A Ca2+-free external medium moderately decreased, whereas a depletion of the intracellular Ca2+ storage sites by cyclopiazonic acid markedly depressed the [Ca2+]i transients induced by either ATP or UTP. Further, the P2Y1 receptor antagonistic PPADS and MRS 2179, as well as the nucleotide catalyzing enzyme apyrase, allmost abolished the effects of these two nucleotides. However, the P2Y1,2,12 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 P2Y1,2,4-immunoreactivities (IR) with nestin-IR at these cells. In conclusion, UTP may induce the release of ATP from hmNPCs via P2Y2 receptor-activation and thereby causes [Ca2+]i transients by stimulating a P2Y1-like receptor. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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]

Abstract: 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]. [Copyright &y& Elsevier]

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]

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. [Copyright &y& Elsevier]

The effects of the adenosine A2A 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 Mg2+-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 A2A 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 α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-induced currents were not affected. The results suggest that adenosine A2A receptor agonists inhibit the NMDA component of the EPSC. [Copyright &y& Elsevier]

Uridine 5′-diphosphoglucose (UDP-G) as a preferential agonist, but also other UDP-sugars, such as UDP galactose, function as extracellular signaling molecules under conditions of cell injury and apoptosis. Consequently, UDP-G is regarded to function as a damage-associated molecular pattern (DAMP), regulating immune responses. UDP-G promotes neutrophil recruitment, leading to the release of pro-inflammatory chemokines. As a potent endogenous agonist with the highest affinity for the P2Y 14 receptor (R), it accomplishes an exclusive relationship between P2Y 14 Rs in regulating inflammation via cyclic adenosine monophosphate (cAMP), nod-like receptor protein 3 (NLRP3) inflammasome, mitogen-activated protein kinases (MAPKs), and signal transducer and activator of transcription 1 (STAT1) pathways. In this review, we initially present a brief introduction into the expression and function of P2Y 14 Rs in combination with UDP-G. Subsequently, we summarize emerging roles of UDP-G/P2Y 14 R signaling pathways that modulate inflammatory responses in diverse systems, and discuss the underlying mechanisms of P2Y 14 R activation in inflammation-related diseases. Moreover, we also refer to the applications as well as effects of novel agonists/antagonists of P2Y 14 Rs in inflammatory conditions. In conclusion, due to the role of the P2Y 14 R in the immune system and inflammatory pathways, it may represent a novel target for anti-inflammatory therapy. • UDP-G/P2Y14R signaling pathways modulate inflammatory responses. • P2Y14R functions as a proinflammatory mediator. Antagonism of P2Y 14 R may be a promising treatment for inflammation-related diseases [ABSTRACT FROM AUTHOR]

• 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]

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. [ABSTRACT FROM AUTHOR]

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]

The aim of the present work was to clarify whether heterotrimeric P2X2/3 receptors have a fixed subunit stoichiometry consisting of one P2X2 and two P2X3 subunits as previously suggested, or a flexible stoichiometry containing also the inverse subunit composition. For this purpose we transfected HEK293 cells with P2X2 and P2X3 encoding cDNA at the ratios of 1:2 and 4:1, and analysed the biophysical and pharmacological properties of the generated receptors by means of the whole-cell patch-clamp technique. The concentration-response curves for the selective agonist α,β-meATP did not differ from each other under the two transfection ratios. However, co-expression of an inactive P2X2 mutant and the wild type P2X3 subunit and vice versa resulted in characteristic distortions of the α,β-meATP concentration–response relationships, depending on which subunit was expressed in excess, suggesting that HEK293 cells express mixtures of (P2X2) 1 /(P2X3) 2 and (P2X2) 2 /(P2X3) 1 receptors. Whereas the allosteric modulators H + and Zn 2+ failed to discriminate between the two possible heterotrimeric receptor variants, the α,β-meATP-induced responses were blocked more potently by the competitive antagonist A317491, when the P2X2 subunit was expressed in deficit of the P2X3 subunit. Furthermore, blue-native PAGE analysis of P2X2 and P2X3 subunits co-expressed in Xenopus laevis oocytes and HEK293 cells revealed that plasma membrane-bound P2X2/3 receptors appeared in two clearly distinct heterotrimeric complexes: a (P2X2-GFP) 2 /(P2X3) 1 complex and a (P2X2-GFP) 1 /(P2X3) 2 complex. These data strongly indicate that the stoichiometry of the heteromeric P2X2/3 receptor is not fixed, but determined in a permutational manner by the relative availability of P2X2 and P2X3 subunits. [ABSTRACT FROM AUTHOR]

Highlights: [•] VTA/SN-PFC organotypic co-cultures show cellular-specific susceptibility to OGD. [•] Neurons are mostly affected in parallel with a marked microglial activation. [•] The P2 antagonist PPADS and valproic acid protect organotypic cultures from OGD. [ABSTRACT FROM AUTHOR]

Abstract: Neurogenesis requires the balance between the proliferation of newly formed progenitor cells and subsequent death of surplus cells. RT-PCR and immunocytochemistry demonstrated the presence of P2X7 receptor mRNA and immunoreactivity in cultured neural progenitor cells (NPCs) prepared from the adult mouse subventricular zone (SVZ). Whole-cell patch-clamp recordings showed a marked potentiation of the inward current responses both to ATP and the prototypic P2X7 receptor agonist dibenzoyl-ATP (Bz-ATP) at low Ca2+ and zero Mg2+ concentrations in the bath medium. The Bz-ATP-induced currents reversed their polarity near 0 mV; in NPCs prepared from P2X7−/− mice, Bz-ATP failed to elicit membrane currents. The general P2X/P2Y receptor antagonist PPADS and the P2X7 selective antagonists Brilliant Blue G and A-438079 strongly depressed the effect of Bz-ATP. Long-lasting application of Bz-ATP induced an initial current, which slowly increased to a steady-state response. In combination with the determination of YO-PRO uptake, these experiments suggest the dilation of a receptor-channel and/or the recruitment of a dye-uptake pathway. Ca2+-imaging by means of Fura-2 revealed that in a Mg2+-deficient bath medium Bz-ATP causes [Ca2+]i transients fully depending on the presence of external Ca2+. The MTT test indicated a concentration-dependent decrease in cell viability by Bz-ATP treatment. Correspondingly, Bz-ATP led to an increase in active caspase 3 immunoreactivity, indicating a P2X7-controlled apoptosis. In acute SVZ brain slices of transgenic Tg(nestin/EGFP) mice, patch-clamp recordings identified P2X7 receptors at NPCs with pharmacological properties identical to those of their cultured counterparts. We suggest that the apoptotic/necrotic P2X7 receptors at NPCs may be of particular relevance during pathological conditions which lead to increased ATP release and thus could counterbalance the ensuing excessive cell proliferation. [Copyright &y& Elsevier]

Abstract: Current responses from CA1 neurons and stratum oriens astrocytes were recorded from hippocampal brain slices by means of the whole-cell patch-clamp technique. Anoxic depolarization (AD) was induced by an oxygen/glucose-deprived (OGD) medium also containing sodium iodoacetate and antimycin, in order to block glycolysis and oxidative phosphorylation, respectively. Anoxic depolarization has been reported to be due to the sudden increase of the extracellular K+ concentration and the accompanying explosive rise in glutamate concentration. We asked ourselves whether the release of ATP activating P2X7 receptors is also involved in the AD. Although, the AD was evoked in absolute synchrony in neurons and astrocytes, and the NMDA receptor antagonistic AP-5 depressed these responses, neither the non-selective P2 receptor antagonist PPADS, nor the highly selective P2X7 receptor antagonist A438079 interfered with the AD or its delay time in neurons/astrocytes after inducing chemical hypoxia. However, A438079, but not PPADS increased in astrocytes the slow inward current observed in a hypoxic medium. It is concluded that ATP co-released with glutamate by hypoxic stimulation has only a minor function in the present brain slice system. [Copyright &y& Elsevier]

The aim of the present experiments was to clarify the subunit stoichiometry of P2X2/3 and P2X2/6 receptors, where the same subunit (P2X2) forms a receptor with two different partners (P2X3 or P2X6). For this purpose, four non-functional Ala mutants of the P2X2, P2X3, and P2X6 subunits were generated by replacing single, homologous amino acids particularly important for agonist binding. Co-expression of these mutants in HEK293 cells to yield the P2X2 WT/P2X3 mutant or P2X2 mutant/P2X3 WT receptors resulted in a selective blockade of agonist responses in the former combination only. In contrast, of the P2X2 WT/P2X6 mutant and P2X2 mutant/P2X6 WT receptors, only the latter combination failed to respond to agonists. The effects of α, β-methylene-ATP and 2-methylthio- ATP were determined by measuring transmembrane currents by the patch clamp technique and intracellular Ca2+ transients by the Ca2+-imaging method. Protein labeling, purification, and PAGE confirmed the assembly and surface trafficking of the investigated WT and WT/mutant combinations in Xenopus laevis oocytes. In conclusion, both electrophysiological and biochemical investigations uniformly indicate that one subunit of P2X2 and two subunits of P2X3 form P2X2/3 heteromeric receptors, whereas two subunits of P2X2 and one subunit of P2X6 constitute P2X2/6 receptors. Further, it was shown that already two binding sites of the three possible ones are sufficient to allow these receptors to react with their agonists. [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]

P2X7 receptors have emerged as potential drug targets for the treatment of medical conditions such as e.g. rheumatoid arthritis and neuropathic pain. To assess the impact of pharmaceuticals on P2X7, we screened a compound library comprising approved or clinically tested drugs and identified several compounds that augment the ATP-triggered P2X7 activity in a stably transfected HEK293 cell line. Of these, clemastine markedly sensitized Ca2+ entry through P2X7 to lower ATP concentrations. Extracellularly but not intracellularly applied clemastine rapidly and reversibly augmented P2X7-mediated whole-cell currents evoked by non-saturating ATP concentrations. Clemastine also accelerated the ATP-induced pore formation and Yo-Pro-1 uptake, increased the fractional NMDG+ permeability, and stabilized the open channel conformation of P2X7. Thus, clemastine is an extracellularly binding allosteric modulator of P2X7 that sensitizes P2X7 to lower ATP concentrations and facilitates its pore dilation. The activity of clemastine on native P2X7 receptors, Ca2+ entry, and whole-cell currents was confirmed in human monocyte-derived macrophages. Similar effects were observed in murine bone marrow-derived macrophages. Consistent with the data on recombinant P2X7, clemastine augmented the ATP-induced cation entry and Yo-Pro-1 uptake. In accordance with the observation that P2X7 controls the cytokine release from LPS-primed macrophages, we found that clemastine augmented the IL-1β release from LPS-primed human macrophages. Collectively, these data point to a sensitization of the recombinantly or natively expressed human P2X7 receptor toward its physiological activator, ATP, possibly leading to a modulation of macrophage-dependent immune responses. [ABSTRACT FROM AUTHOR]

Homomeric P2X3 receptors are present in sensory ganglia and participate in pain perception. Amino acid (AA) residues were replaced in the four supposed nucleotide binding segments (NBSs) of the human (h) P2X3 receptor by alanine, and these mutants were expressed in HEK293 cells and Xenopus laevis oocytes. Patch clamp and two-electrode voltage clamp measurements as well as the Ca2+ imaging technique were used to compare the concentration-response curves of the selective P2X1,3 agonist α,β-methylene ATP obtained at the wild-type P2X3 receptor and its NBS mutants. Within these NBSs, certain Gly (Gly-66), Lys (Lys-63, Lys-176, Lys-284, Lys-299), Asn (Asn-177, Asn-279), Arg (Arg-281, Arg-295), and Thr (Thr-172) residues were of great importance for a full agonist response. However, the replacement of further AAs in the NBSs by Ala also appeared to modify the amplitude of the current and/or [Ca2+]i responses, although sometimes to a minor degree. The agonist potency decrease was additive after the simultaneous replacement of two adjacent AAs by Ala (K65A/G66A, F171A/T172A, N279A/F280A, F280A/R281A) but was not altered after Ala substitution of two non-adjacent AAs within the same NBS (F171A/N177A). SDS-PAGE in the Cy5 cell surface-labeled form demonstrated that the mutants appeared at the cell surface in oocytes. Thus, groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the hP2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor. [ABSTRACT FROM AUTHOR]

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]