Characterization of cultured dorsal root ganglion neuron P2X receptors

P2X receptors for adenosine 5′-triphosphate (ATP) comprise a family of ligand-gated cation channels with distinct characteristics which are dependent on the receptor subunits (P2X_1–7) expressed, and the homomeric or heteromeric assembly of protein subunits in individual cells. We describe the properties of P2X receptors expressed by cultured adult rat dorsal root ganglion cells on the basis of the time course of responses to ATP, α,β-methylene adenosine 5′-triphosphate (α,β-meATP) and 2-methyl-thioadenosine 5′-triphosphate (2-meSATP), and using the antagonists 2′,3′-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), a novel and highly selective purinoceptor antagonist, suramin and iso-pyridocalphosphate-6-azophenyl-2′,5′ disulphonic acid (PPADS). ATP (10 μm ) evoked inward currents in ≈ 95% of neurons tested and > 80% responded with a fast transient inward current that rapidly inactivated during the continued presence of ATP. Of the remaining neurons, ≈ 4% showed a sustained response and ≈ 10% showed a combination of transient and sustained components. Rapid application of ATP, α,β-meATP and 2meSATP demonstrated these to be full agonists of the rapidly inactivating P2X receptor (pA₅₀ values = 5.83, 5.86 and 5.55, respectively), whilst uridine triphosphate (UTP) and 1-β,γ-methyleneadenosine 5′-triphosphate (1-β,γ-meATP) were ineffective as agonists. These rapidly inactivating responses could be inhibited by TNP-ATP, suramin and PPADS (pIC₅₀ = 9.5, 6.5, 6.4, respectively). Using inactivation protocols, we demonstrate the presence of homomeric P2X₃-like receptors and non-inactivating P2X receptors, which indicates that individual subsets of adult dorsal root ganglion neurons have distinct P2X receptor phenotypes, and that individual DRG neurons may express multiple P2X receptor subtypes.     

Electroacupuncture and A‐317491 depress the transmission of pain on primary afferent mediated by the P2X3 receptor in rats with chronic neuropathic pain states

P2X is a family of ligand‐gated ion channels that act through adenosine ATP. The P2X₃ receptor plays a key role in the transmission of neuropathic pain at peripheral and spinal sites. Electroacupuncture (EA) has been used to treat neuropathic pain effectively. To determine the role of EA in neuropathic pain mediated through the P2X₃ receptor in dorsal root ganglion neurons and the spinal cord, a chronic constriction injury (CCI) model was used. Sprague‐Dawley rats were divided into four groups: sham CCI, CCI, CCI plus contralateral EA, and CCI plus ipsilateral EA. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were recorded. Furthermore, the expression of the P2X₃ receptor was evaluated through Western blotting and immunofluorescence. The effects of EA and A‐317491 were investigated through the whole‐cell patch‐clamp method and intrathecal administration. Our results show that the MWT and TWL of EA groups were higher than those in the CCI group, whereas the expression of the P2X₃ receptor was lower than that in the CCI group. However, no significant difference was detected between the two EA groups. EA depressed the currents created by ATP and the upregulation of the P2X₃ receptor in CCI rats. Additionally, EA was more potent in reducing mechanical allodynia and thermal hyperalgesia when combined with A‐317491 through intrathecal administration. These results show that both contralateral and ipsilateral EA might inhibit the primary afferent transmission of neuropathic pain induced through the P2X₃ receptor. In addition, EA and A‐317491 might have an additive effect in inhibiting the transmission of pain mediated by the P2X₃ receptor. © 2014 Wiley Periodicals, Inc.     

P2X7R‐mediated Ca2+‐independent d‐serine release via pannexin‐1 of the P2X7R‐pannexin‐1 complex in astrocytes

d ‐serine is a coagonist of N‐methyl‐d ‐aspartate (NMDA) subtype of glutamate receptor and plays a role in regulating activity‐dependent synaptic plasticity. In this study, we examined the mechanism by which extracellular ATP triggers the release of d ‐serine from astrocytes and discovered a novel Ca²⁺‐independent release mechanism mediated by P2X₇ receptors (P2X₇R). Using [³H] d ‐serine, which was loaded into astrocytes via the neutral amino acid transporter 2 (ASCT2), we observed that ATP and a potent P2X₇R agonist, 2′(3′)‐O‐(4‐benzoylbenzoyl)adenosine‐5′‐triphosphate (BzATP), stimulated [³H]D‐serine release and that were abolished by P2X₇R selective antagonists and by shRNAs, whereas enhanced by removal of intracellular or extracellular Ca²⁺. The P2X₇R‐mediated d ‐serine release was inhibited by pannexin‐1 antagonists, such as carbenoxolone (CBX), probenecid (PBN), and ¹⁰Panx‐1 peptide, and shRNAs, and stimulation of P2X₇R induced P2X₇R‐pannexin‐1 complex formation. Simply incubating astrocytes in Ca²⁺/Mg²⁺‐free buffer also induced the complex formation, and that enhanced basal d ‐serine release through pannexin‐1. The P2X₇R‐mediated d ‐serine release assayed in Ca²⁺/Mg²⁺‐free buffer was enhanced as well, and that was inhibited by CBX. Treating astrocytes with general protein kinase C (PKC) inhibitors, such as chelerythrine, GF109203X, and staurosporine, but not Ca²⁺‐dependent PKC inhibitor, Gö6976, inhibited the P2X₇R‐mediated d ‐serine release. Thus, we conclude that in astrocytes, P2X₇R‐pannexin‐1 complex formation is crucial for P2X₇R‐mediated d ‐serine release through pannexin‐1 hemichannel. The release is Ca²⁺‐independent and regulates by a Ca²⁺‐independent PKC. The activated P2X₇R per se is also functioned as a permeation channel to release d ‐serine in part. This P2X₇R‐mediated d ‐serine release represents an important mechanism for activity‐dependent neuron‐glia interaction. GLIA 2015;63:877–893     

Expressions of P2X2 and P2X3 receptors in rat nodose neurons after myocardial ischemia injury

The role of ATP is as a functional neurotransmitter and local intercellular signaling molecule. The nodose neurons express both P2X₂ and P2X₃ subunits in their plasma membrane. This study wants to observe the expression of P2X₂ receptor and the expression relationship between P2X₂ and P2X₃ in nodose neurons after myocardial ischemic injury. The expressions of P2X₃ immunoreactivity, mRNA and protein were analyzed by immunohistochemistry, in situ hybridization and western blotting. P2X₂ and P2X₃ immunoreactivity, mRNA expression had been increased after myocardial ischemia in nodose neurons. Myocardial ischemia enhanced P2X₂ and P2X₃ protein level in nodose ganglia after myocardial ischemia. P2X₂ receptor in nodose neurons participated in the transmission of cardiac pain. The changes of P2X₂ and P2X₃ immunoreactivities, mRNA and protein that occurred following myocardial ischemic injury in the nodose ganglia showed that a correlation existed between P2X₂ and P2X₃ expression. It suggests that P2X₂ receptor subtype in company with P2X₃ receptor subtype plays the important role in cardiac vagal sensory nociceptors with a sensitivity to ATP.     

P2X4 receptor in the dorsal horn partially contributes to brain‐derived neurotrophic factor oversecretion and toll‐like receptor‐4 receptor activation associated with bone cancer pain

Previous studies have suggested that the microglial P2X7 purinoceptor is involved in the release of tumor necrosis factor‐α (TNFα) following activation of toll‐like receptor‐4 (TLR4), which is associated with nociceptive behavior. In addition, this progress is evoked by the activation of the P2X4 purinoceptor (P2X4R). Although P2X4R is also localized within spinal microglia in the dorsal horn, little is known about its role in cancer‐induced bone pain (CIBP), which is in some ways unique. With the present rat model of CIBP, we demonstrate a critical role of the microglial P2X4R in the enhanced nociceptive transmission, which is associated with TLR4 activation and secretion of brain‐derived neurotrophic factor (BDNF) and TNFα in the dorsal horn. We assessed mechanical threshold and spontaneous pain of CIBP rats. Moreover, P2X4R small interfering RNA (siRNA) was administered intrathecally, and real‐time PCR, Western blots, immunofluorescence histochemistry, and ELISA were used to detect the expression of P2X4R, TLR4, OX‐42, phosphorylated‐p38 MAPK (p‐p38), BDNF, and TNFα. Compared with controls, intrathecal injection of P2X4R siRNA could prevent nociceptive behavior induced by ATP plus lipopolysaccharide and CIBP and reduce the expression of P2X4R, TLR4, p‐p38, BDNF, and TNFα. In addition, the increase of BDNF protein in rat microglial cells depended on P2X4 receptor signaling, which is partially associated with TLR4 activation. The ability of microglial P2X4R to activate TLR4 in spinal cord leading to behavioral hypersensitivity and oversecretion of BDNF could provide an opportunity for the prevention and treatment of CIBP. © 2014 Wiley Periodicals, Inc.     

Increase of intracellular Ca2+ by P2Y but not P2X receptors in cultured cortical multipolar neurons of the rat

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. P2X₁ and P2X₇ 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), ADPβS, 2MeSADP, or 2MeSATP and even fewer cells to UTP. In contrast, α,β‐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‐N⁶‐methyl adenosine 3′,5′‐diphosphate (MRS2179), and suramin (300 μM) 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 ADPβS 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 P2X₇, remains elusive. J. Comp. Neurol. 516:343–359, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of puerarin on P2X3 receptor involved in hyperalgesia after burn injury in the rat

The study investigated the effects of puerarin on P2X₃ receptor involved in hyperalgesia after burn injury in the rat. Superficial second degree burn injury models were adopted. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured and the P2X₃ receptor expressions in dorsal root ganglion (DRG) from burn injury models rats were detected by immunohistochemistry, in situ hybridization, RT-PCR and western blot. MWL and TWL in untreated superficial second paw burn rats were reduced. MWL and TWL of puerarin-treated superficial second paw burn rats showed significant increase compared with untreated superficial second paw burn rats. Puerarin can decrease the hyperalgesia after burn injury. At day 3 post-burn, the expressions of P2X₃ protein and mRNA in DRG neurons in untreated superficial second degree back burn group were increased significantly compared with sham back burn group, puerarin-treated back unburned control group, blank back control group, while in puerarin-treated superficial second degree back burn group, the P2X₃ protein and mRNA expressions were decreased markedly. There is no significant difference in sham back burn group, puerarin-treated back unburned control group, blank back control group. Therefore, puerarin may reduce the nociceptive transmission of burn injury pain mediated by P2X₃ receptor and alleviate P2X₃ receptor involved in hyperalgesia after burn injury in the rats.     

Astrocyte–neuron interaction in the substantia gelatinosa of the spinal cord dorsal horn via P2X7 receptor‐mediated release of glutamate and reactive oxygen species

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 Ca²⁺/no Mg²⁺ (low X²⁺) external medium adenosine triphosphate (ATP)/dibenzoyl‐ATP, Bz‐ATP) caused inward current responses, much larger in amplitude than those recorded in a normal X²⁺‐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 H₂O₂ 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, H₂O₂ 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. GLIA 2014;62:1671–1686     

Neurite outgrowth of striatal neurons in vitro: involvement of purines in the growth-promoting effect of myenteric plexus explants

We have shown previously that a soluble factor(s) released by the myenteric plexus promotes neurite outgrowth from postnatal striatal neurons, and that this effect was abolished by tetrodotoxin. We have now investigated the possible involvement of purines in the mediation of this neuritogenic response, by examining their effect on neurite length of striatal neurons both in co-culture with myenteric plexus explants and cultured alone.Both ATP and 2-chloroadenosine partially reversed the inhibitory effect of tetrodotoxin in co-cultures with whole myenteric plexus, while the stable ATP analogue, α,β-methylene ATP, had no effect, suggesting that ATP was being broken down to adenosine before exerting its action. Further support for this view was that the ATP (P2) purinoceptor antagonist suramin did not reverse the effects of ATP, while the adenosine (P1) purinoceptor antagonist 8-(p-sulphophenyl)theophylline did antagonize the effects of ATP in tetrodotoxin-treated co-cultures. Further, both 8-(p-sulphophenyl)theophylline and adenosine deaminase reduced the effect of the myenteric plexus on striatal neurons in the absence of tetrodotoxin, and the adenylate cyclase activator forskolin completely reversed the effect of tetrodotoxin in our co-culture system.The neurite outgrowth-promoting effect of 2-chloroadenosine in tetrodotoxin-treated co-cultures was not further enhanced by a combination of neuropeptides. Serotonin and GTP were without effect on striatal neurons in the presence or absence of myenteric plexus explants. In experiments without myenteric plexus, both 2-chloroadenosine and forskolin caused a slight increase in striatal neurite length; ATP and GTP were ineffective. Basic fibroblast growth factor, nerve growth factor, neurotrophin-3 or neurotrophin-4/5 had no effect on neurite outgrowth in postnatal striatal cultures after two days in vitro. When these growth factors were added in combination with 2-chloroadenosine, the observed increase in mean neurite length did not exceed that induced by 2-chloroadenosine alone. Both 2-chloroadenosine and the ganglioside mix AGF1 increased neurite elongation of striatal neurons after two days in vitro, but an inhibition of enhanced neurite outgrowth was observed when both substances were added together. Both laminin and fibronectin were not neuritogenic for postnatal striatal neurons under our culture conditions. These observations suggest that a factor other than the growth factors tested here is involved in the promotion of striatal neurite outgrowth in co-culture with myenteric plexus explants.In summary, adenosine (probably acting through the A₂ subclass of the P1 purinoceptor) leads to increased striatal neurite outgrowth in co-culture with myenteric plexus and we propose that it does so either (1) by triggering the release of a neuritogenic factor, possibly from enteric glial cells, or (2) by acting synergistically with such a growth factor. Adenosine acts via PI purinoceptors, which leads to changes in cyclic AMP, and the response to forskolin suggests that cyclic AMP is probably involved in the events leading to increased striatal neurite outgrowth.     

Ca2+‐dependent release of ATP from astrocytes affects herpes simplex virus type 1 infection of neurons

Astrocytes provide metabolic support for neurons and modulate their functions by releasing a plethora of neuroactive molecules diffusing to neighboring cells. Here we report that astrocytes also play a role in cortical neurons' vulnerability to Herpes simplex virus type‐1 (HSV‐1) infection through the release of extracellular ATP. We found that the interaction of HSV‐1 with heparan sulfate proteoglycans expressed on the plasma membrane of astrocytes triggered phospholipase C‐mediated IP₃‐dependent intracellular Ca²⁺ transients causing extracellular release of ATP. ATP binds membrane purinergic P2 receptors (P2Rs) of both neurons and astrocytes causing an increase in intracellular Ca²⁺ concentration that activates the Glycogen Synthase Kinase (GSK)‐3β, whose action is necessary for HSV‐1 entry/replication in these cells. Indeed, in co‐cultures of neurons and astrocytes HSV‐1‐infected neurons were only found in proximity of infected astrocytes releasing ATP, whereas in the presence of fluorocitrate, an inhibitor of astrocyte metabolism, switching‐off the HSV‐1‐induced ATP release, very few neurons were infected. The addition of exogenous ATP, mimicking that released by astrocytes after HSV‐1 challenge, restored the ability of HSV‐1 to infect neurons co‐cultured with metabolically‐inhibited astrocytes. The ATP‐activated, P2R‐mediated, and GSK‐3‐dependent molecular pathway underlying HSV‐1 infection is likely shared by neurons and astrocytes, given that the blockade of either P2Rs or GSK‐3 activation inhibited infection of both cell types. These results add a new layer of information to our understanding of the critical role played by astrocytes in regulating neuronal functions and their response to noxious stimuli including microbial agents via Ca²⁺‐dependent release of neuroactive molecules.     

ATP increases extracellular dopamine level through stimulation of P2Y purinoceptors in the rat striatum

The effect of ATP on release of dopamine (DA) from rat striatum was studied using in vivo microdialysis. ATP increased the striatal extracellular levels of DA dose-dependently. These analogs produced an increase in DA according to this order of potency: 2-methylthio ATP > ATP α, β-methylene ATP > ADP > AMP > adenosine. Adenosine 5′-[β, γ imido]-triphosphate had a more prolonged effect on the increase in DA level than ATP. The ATP-induced increase in DA was inhibited by adding suramin, a nonselective P₂ purinoceptor antagonist, and reactive blue 2, a P_2Y purinoceptor antagonist, but not inhibited by xanthine amine congener, an adenosine receptor antagonist. Pertussis toxin reduced the increase in DA produced by ATP, whih suggests that te P₂ purinoceptor may be coupled with a G-protein in the rat striatum. Results suggest that P_2Y purinoceptors may involve an ATP-induced increase in DA. The ATP-induced release of DA was tetrodotoxin-sensitive, Ca²⁺-dependent and was abolished by ω-conotoxin GVIA, indicating that the opening of voltage-sensitive Na⁺ channel and the Ca²⁺ influx through the N-type voltage-dependent calcium channel are both required for the ATP-induced increase in DA. The ATP-induced increase in DA is presumably due to the release of DA via the stimulation of P_2Y purinoceptors in the rat striatum.     

Microglia‐derived purines modulate mossy fibre synaptic transmission and plasticity through P2X4 and A1 receptors

Recent data have provided evidence that microglia, the brain‐resident macrophage‐like cells, modulate neuronal activity in both physiological and pathophysiological conditions, and microglia are therefore now recognized as synaptic partners. Among different neuromodulators, purines, which are produced and released by microglia, have emerged as promising candidates to mediate interactions between microglia and synapses. The cellular effects of purines are mediated through a large family of receptors for adenosine and for ATP (P2 receptors). These receptors are present at brain synapses, but it is unknown whether they can respond to microglia‐derived purines to modulate synaptic transmission and plasticity. Here, we used a simple model of adding immune‐challenged microglia to mouse hippocampal slices to investigate their impact on synaptic transmission and plasticity at hippocampal mossy fibre (MF) synapses onto CA3 pyramidal neurons. MF–CA3 synapses show prominent forms of presynaptic plasticity that are involved in the encoding and retrieval of memory. We demonstrate that microglia‐derived ATP differentially modulates synaptic transmission and short‐term plasticity at MF–CA3 synapses by acting, respectively, on presynaptic P2X₄ receptors and on adenosine A₁ receptors after conversion of extracellular ATP to adenosine. We also report that P2X₄ receptors are densely located in the mossy fibre tract in the dentate gyrus–CA3 circuitry. In conclusion, this study reveals an interplay between microglia‐derived purines and MF–CA3 synapses, and highlights microglia as potent modulators of presynaptic plasticity.     

Molecular mechanisms of cross‐inhibition between nicotinic acetylcholine receptors and P2X receptors in myenteric neurons and HEK‐293 cells

Background P2X₂ and nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic excitation in the enteric nervous system. P2X receptors and nAChRs are functionally linked. This study examined the mechanisms responsible for interactions between P2X2 and α3β4subunit‐containing nAChRs. Methods The function of P2X2 and α3β4 nAChRs expressed by HEK‐293 cells and guinea pig ileum myenteric neurons in culture was studied using whole‐cell patch clamp techniques. Key Results In HEK‐293 cells expressing α3β4 nAChRs and P2X2 receptors, co‐application of ATP and acetylcholine caused inward currents that were 56 ± 7% of the current that should occur if these channels functioned independently (P < 0.05, n = 9); we call this interaction cross‐inhibition. Cross‐inhibition did not occur in HEK‐293 cells expressing α3β4 nAChRs and a C‐terminal tail truncated P2X2 receptor (P2X2TR) (P > 0.05, n = 8). Intracellular application of the C‐terminal tail of the P2X2 receptor blocked nAChR‐P2X receptor cross‐inhibition in HEK‐293 cells and myenteric neurons. In the absence of ATP, P2X2 receptors constitutively inhibited nAChR currents in HEK‐293 cells expressing both receptors. Constitutive inhibition did not occur in HEK‐293 cells expressing α3β4 nAChRs transfected with P2X2TR. Currents caused by low (≤30 μmol L^?1), but not high (≥100 μmol L^?1) concentrations of ATP in cells expressing P2X2 receptors were inhibited by co‐expression with α3β4 nAChRs. Conclusions & Inferences The C‐terminal tail of P2X2 receptors mediates cross‐inhibition between α3β4 nAChR‐P2X2 receptors. The closed state of P2X2 receptors and nAChRs can also cause cross‐inhibition. These interactions may modulate transmission at enteric synapses that use ATP and acetylcholine as co‐transmitters.     

Reciprocal regulation of ATPγS‐induced monocyte chemoattractant protein‐1 production by ERK and p38 MAP kinases in rat corticostriatal slice cultures

Monocyte chemoattractant protein‐1 (MCP‐1, CCL2) is a well‐defined chemokine implicated in the pathology of various neurodegenerative diseases and brain injuries, such as Alzheimer's disease, multiple sclerosis, stroke, and traumatic injury. We investigated the effect of the activation of P2 purinoceptors on MCP‐1 production in rat corticostriatal slice cultures. Treatment with adenosine 5′‐O‐(3‐thiotriphosphate) (ATPγS), a hydrolysis‐resistant adenosine triphosphate (ATP) analog, induced MCP‐1 production in astrocytes. The induction was in a concentration‐dependent manner and was antagonized by a P2 purinoceptor antagonist pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid. The inhibition of an extracellular signal‐regulated kinase (ERK) pathway by PD98059 and U0126 significantly suppressed ATPγS‐induced MCP‐1 mRNA expression and protein production, while inhibition of c‐Jun N‐terminal kinase by SP600125 resulted in the partial suppression. Conversely, SB203580, a p38 mitogen‐activated protein (MAP) kinase inhibitor, significantly enhanced ATPγS‐induced MCP‐1 production. Similar effects of ERK and p38 MAP kinase inhibitors on MCP‐1 production were observed in the slices stimulated by ATP and BzATP. These results demonstrate that astrocytic MCP‐1 production induced by P2 purinoceptor stimulation is reciprocally regulated by ERK and p38 MAP kinases in the organotypic slice cultures. © 2009 Wiley‐Liss, Inc.     

P2 receptor‐mediated stimulation of the PI3‐K/Akt‐pathway in vivo

ATP acts as a growth factor as well as a toxic agent by stimulating P2 receptors. The P2 receptor‐activated signaling cascades mediating cellular growth and cell survival after injury are only incompletely understood. Therefore, the aim of the present study was to identify the role of the phosphoinositide 3 kinase (PI3‐K/Akt) and the mitogen‐activated protein kinase/extracellular signal regulated protein kinase (MAPK/ERK) pathways in P2Y receptor‐mediated astrogliosis after traumatic injury and after microinfusion of ADPβS (P2Y_1,12,13 receptor agonist) into the rat nucleus accumbens (NAc). Mechanical damage and even more the concomitant treatment with ADPβS, enhanced P2Y₁ receptor‐expression in the NAc, which could be reduced by pretreatment with the P2X/Y receptor antagonist PPADS. Quantitative Western blot analysis indicated a significant increase in phosphorylated (p)Akt and pERK1/2 2 h after ADPβS‐microinjection. Pretreatment with PPADS or wortmannin abolished the up‐regulation of pAkt by injury alone or ADPβS‐treatment. The ADPβS‐enhanced expression of the early apoptosis marker active caspase 3 was reduced by PPADS and PD98059, but not by wortmannin. Multiple immunofluorescence labeling indicated a time‐dependent expression of pAkt and pMAPK on astrocytes and neurons and additionally the colocalization of pAkt, pMAPK, and active caspase 3 with the P2Y₁ receptor especially at astrocytes. In conclusion, the data show for the first time the involvement of PI3‐K/Akt‐pathway in processes of injury‐induced astroglial proliferation and anti‐apoptosis via activation of P2Y₁ receptors in vivo, suggesting specific roles of P2 receptors in glial cell pathophysiology in neurodegenerative diseases. © 2008 Wiley‐Liss, Inc.     

P2X7‐dependent,but differentially regulated release of IL‐6, CCL2, and TNF‐α in cultured mouse microglia

ATP is an important regulator of microglia and its effects on microglial cytokine release are currently discussed as important contributors in a variety of brain diseases. We here analyzed the effects of ATP on the production of six inflammatory mediators (IL‐6, IL‐10, CCL2, IFN‐γ, TNF‐α, and IL‐12p70) in cultured mouse primary microglia. Stimulation of P2X7 receptor by ATP (1 mM) or BzATP (500 µM) evoked the mRNA expression and release of proinflammatory cytokines IL‐6, TNF‐α, and the chemokine CCL2 in WT cells but not in P2X7^?/? cells. The effects of ATP and BzATP were inhibited by the nonselective P2 receptor antagonists PPADs and suramin. Various selective P2X7 receptor antagonists blocked the P2X7‐dependent release of IL‐6 and CCL2, but, surprisingly, had no effect on BzATP‐induced release of TNF‐α in microglia. Calcium measurements confirmed that P2X7 is the main purine receptor activated by BzATP in microglia and showed that all P2X7 antagonists were functional. It is also presented that pannexin‐1 hemichannel function and potential P2X4/P2X7 heterodimers are not involved in P2X7‐dependent release of IL‐6, CCL2, and TNF‐α in microglia. How P2X7‐specific antagonists only affect P2X7‐dependent IL‐6 and CCL2 release, but not TNF‐α release is at the moment unclear, but indicates that the P2X7‐dependent release of cytokines in microglia is differentially regulated. GLIA 2014;62:592–607     

The neural crest‐ and placodes‐derived afferent innervation of the mouse esophagus

Background The mouse is an invaluable model for mechanistic studies of esophageal nerves, but the afferent innervation of the mouse esophagus is incompletely understood. Vagal afferent neurons are derived from two embryonic sources: neural crest and epibranchial placodes. We hypothesized that both neural crest and placodes contribute to the TRPV1‐positive (potentially nociceptive) vagal innervation of the mouse esophagus. Methods Vagal jugular/nodose ganglion (JNG) and spinal dorsal root ganglia (DRG) neurons were retrogradely labeled from the cervical esophagus. Single cell RT‐PCR was performed on the labeled neurons. Key Results In the Wnt1Cre/R26R mice expressing a reporter in the neural crest‐derived cells we found that both the neural crest‐ and the placodes‐derived vagal JNG neurons innervate the mouse esophagus. In the wild‐type mouse the esophageal vagal JNG TRPV1‐positive neurons segregated into two subsets: putative neural crest‐derived purinergic receptor P2X₂‐negative/preprotachykinin‐A (PPT‐A)‐positive subset and putative placodes‐derived P2X₂‐positive/PPTA‐negative subset. These subsets also segregated by the expression of TrkA and GFRα₃ in the putative neural crest‐derived subset, and TrkB in the putative placodes‐derived subset. The TRPV1‐positive esophageal DRG neurons had the phenotype similar to the vagal putative neural crest‐derived subset. Conclusions & Inferences The TRPV1‐positive (potentially nociceptive) vagal afferent neurons innervating the mouse esophagus originate from both neural crest and placodes. The expression profile of the receptors for neurotrophic factors is similar between the neural crest‐derived vagal and spinal nociceptors, but distinct from the vagal placodes‐derived nociceptors.     

Purinergic signaling in the cerebellum: Bergmann glial cells express functional ionotropic P2X₇ receptors

Astrocytes constitute active networks of intercommunicating cells that support the metabolism and the development of neurons and affect synaptic functions via multiple pathways. ATP is one of the major neurotransmitters mediating signaling between neurons and astrocytes. Potentially acting through both purinergic metabotropic P2Y receptors (P2YRs) and ionotropic P2X receptors (P2XRs), up until now ATP has only been shown to activate P2YRs in Bergmann cells, the radial glia of the cerebellar cortex that envelopes Purkinje cell afferent synapses. In this study, using multiple experimental approaches in acute cerebellar slices we demonstrate the existence of functional P2XRs on Bergmann cells. In particular, we show here that Bergmann cells express uniquely P2X₇R subtypes: (i) immunohistochemical analysis revealed the presence of P2X₇Rs on Bergmann cell processes, (ii) in whole cell recordings P2XR pharmacological agonists induced depolarizing currents that were blocked by specific antagonists of P2X₇Rs, and could not be elicited in slices from P2X₇R‐deficient mice and finally, (iii) calcium imaging experiments revealed two distinct calcium signals triggered by application of exogenous ATP: a transient signal deriving from release of calcium from intracellular stores, and a persistent one following activation of P2X₇Rs. Our data thus reveal a new pathway by which extracellular ATP may affect glial cell function, thus broadening our knowledge on purinergic signaling in the cerebellum. © 2011 Wiley‐Liss, Inc.