Multiple P2Y subtypes in spinal microglia are involved in neuropathic pain after peripheral nerve injury

A prominent signaling pathway in the development of neuropathic pain involves ATP acting on microglial purinergic receptors. Among the P2Y metabotropic receptors, we reported before that the P2Y12 receptor is upregulated in microglia following nerve injury and involved in the phosphorylation of p38 MAPK, and in the development of pain behavior. In this study, we examined the expression of P2Y6, P2Y13, and P2Y14 receptors in the spinal cord and whether these receptors are involved in the pathogenesis of neuropathic pain following peripheral nerve injury. We found that spared nerve injury induced a dramatic increase of not only P2Y12, but also P2Y6, 13, and 14 receptor mRNA expression in spinal microglia. The increase continued for at least 2 weeks after injury. To determine whether p38 MAPK can induce the expression of P2Y receptors, we administered intrathecally the p38 MAPK inhibitor SB203580 and found that it significantly suppressed P2Y6, P2Y13, and P2Y14 but not P2Y12 mRNAs. Intrathecal injection of the specific P2Y6 antagonist MRS2578, specific P2Y13 antagonist MRS2211 or P2Y14 antisense LNA, attenuated mechanical pain hypersensitivity. Themixture of three antagonists for P2Y6, 12, and 13 showed a longer suppressive effect on pain behavior than the individual treatments. Our data demonstrate that ATP and other nucleotides may stimulate activated microglia with the upregulation of P2Y6, P2Y12, P2Y13, and P2Y14 receptors following nerve injury and these receptors are involved in the development of neuropathic pain. © 2012 Wiley Periodicals, Inc.     

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.     

Inhibition of P2X7R in the amygdala ameliorates symptoms of neuropathic pain after spared nerve injury in rats

The amygdala circuitry and P2X7 receptor (P2X7R) have both been shown to play important roles in the modulation of neuropathic pain (NP). However, little is known about the functional role of P2X7R in the amygdala for the regulation of NP. This study aims to evaluate the alleviative effect of intra-amygdala microinfusion of a pharmacological antagonist of P2X7R (A-438079) on NP and explore its possible mechanism of action. Male Sprague-Dawley rats were used to construct the animal model of NP through spared nerve injury (SNI). The SNI rats randomly received chronic bilateral microinjection of A-438079 (100 pmol/side) or saline into the amygdalae via cannulas. Mechanical paw withdrawal threshold (MWT) and thermal withdrawal duration (TWD) were measured by von Frey monofilaments. Besides, tail suspension test (TST), forced swimming test (FST), open field test (OFT) and sucrose preference test (SPT) were performed to assess depression- and anxiety-like behaviors. Immunofluorescence assay was employed to determine the levels of glial fibrillary acidic protein (GFAP), ionized calcium binding adaptor molecule 1 (IBA-1) and connexin 43 (Cx43) in the spinal cord. In addition, the change of growth associated protein 43 (GAP43) level in the spinal cord was assessed by Western blot. Our data showed that chronic treatment with A-438079 increased MWT and decreased TWD on days 11–21 post-SNI while decreased depression-like and anxiety-like behaviors. A-438079 administration significantly attenuated the elevated immunoreactivities of IBA-1 and GFAP in microglia and astrocytes after SNI. Furthermore, the decreased expression of GAP-43 in the spinal cord due to SNI was significantly attenuated by A-438079. However, when A-438079 and a pharmacological agonist (BzATP) of P2X7R were given simultaneously, all the effects caused by A-438079 alone were reversed. In brief, our study revealed the protective role of inhibiting P2X7R in the amygdala against symptoms associated with NP, possibly attributing to its inhibitory effects on spinal microglia and astrocytes.     

Activation of P2X4 receptor exacerbates acute brain injury after intracerebral hemorrhage

IntroductionIntracerebral hemorrhage (ICH) accounts for 10%–15% of all strokes and culminates in high mortality and disability. After ICH, brain injury is initiated by the mass effect of hematoma, followed by secondary cytotoxic injury from dying brain cells, hematoma disintegration, and cascading brain immune response. However, the molecular mechanism of secondary cytotoxic brain injury in ICH is not completely understood. The sensitive purinergic receptor, P2X4 receptor (P2X4R), was known to recognize extracellular free ATP released by dying cells during tissue injury.AimsIn this study, we aim to understand the role of P2X4R in acute brain injury triggered by ICH.ResultsIn this study, we found that the sensitive purinergic receptor, P2X4R, was upregulated in the brain of patients with ICH as well as in a mouse model of ICH induced by collagenase injection. P2X4R blockage with the specific inhibitor 5‐BDBD attenuated brain injury in ICH mice by significantly reducing brain edema, blood–brain barrier leakage, neural death, and ultimately acute neurodeficits. Further study indicated that the protective effect of P2X4R inhibition is related to decreased pro‐inflammatory activity of microglia and recruitment of peripheral immune cells into the hemorrhagic brain.ConclusionsThese results suggest that the P2X4 receptor is activated by ICH stimuli which worsen brain injury following ICH.     

Valproic acid attenuates microgliosis in injured spinal cord and purinergic P2X4 receptor expression in activated microglia

Peripheral injection with a high dose of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, into animals with mild or moderate spinal cord injury (SCI) for 1 week can reduce spinal cord tissue loss and promote hindlimb locomotor recovery. A purinergic adenosine triphosphate (ATP) receptor subtype, P2X₄ receptor (P2X₄R), has been considered as a potential target to diminish SCI‐associated inflammatory responses. In this study, using a minipump‐based infusion system, we found that intraspinal infusion with VPA for 3 days into injured spinal cord significantly improved hindlimb locomotion of rats with severe SCI induced by a 10‐g NYU impactor dropping from the height of 50 mm onto the spinal T9/10 segment. The neuronal fibers in the injured spinal cord tissues were significantly preserved in VPA‐treated rats compared with those observed in vehicle‐treated animals. Moreover, the accumulation of microglia/macrophages and astrocytes in the injured spinal cord was attenuated in the animal group receiving VPA infusion. VPA also significantly reduced P2X₄R expression post‐SCI. Furthermore, in vitro study indicated that VPA, but not the other HDAC inhibitors, sodium butyrate and trichostatin A (TSA), caused downregulation of P2X₄R in microglia activated with lipopolysaccharide (LPS). Moreover, p38 mitogen‐activated protein kinase (MAPK)‐triggered signaling was involved in the effect of VPA on the inhibition of P2X₄R gene expression. In addition to the findings from others, our results also provide important evidence to show the inhibitory effect of VPA on P2X₄R expression in activated microglia, which may contribute to reduction of SCI‐induced gliosis and subsequently preservation of spinal cord tissues. © 2013 Wiley Periodicals, Inc.     

Transforming growth factor-activated kinase 1 induced in spinal astrocytes contributes to mechanical hypersensitivity after nerve injury

Mitogen-activated protein kinase (MAPK) plays an important role in the induction and maintenance of neuropathic pain. Transforming growth factor-activated kinase 1 (TAK1), a member of the MAPK kinase kinase family, is indispensable for the activation of c-Jun N-terminal kinase (JNK) and p38 MAPK. We now show that TAK1 induced in spinal cord astrocytes is crucial for mechanical hypersensitivity after peripheral nerve injury. Nerve injury induced a striking increase in the expression of TAK1 in the ipsilateral dorsal horn, and TAK1 was increased in hyperactive astrocytes, but not in neurons or microglia. Intrathecal administration of TAK1 antisense oligodeoxynucleotide (AS-ODN) prevented and reversed nerve injury-induced mechanical, but not heat hypersensitivity. Furthermore, TAK1 AS-ODN suppressed the activation of JNK1, but not p38 MAPK, in spinal astrocytes. In contrast, there was no change in TAK1 expression in primary sensory neurons, and TAK1 AS-ODN did not attenuate the induction of transient receptor potential ion channel TRPV1 in sensory neurons. Taken together, these results demonstrate that TAK1 upregulation in spinal astrocytes has a substantial role in the development and maintenance of mechanical hypersensitivity through the JNK1 pathway. Thus, preventing the TAK1/JNK1 signaling cascade in astrocytes might provide a fruitful strategy for treating intractable neuropathic pain.     

Spinal P2X(7) receptor mediates microglia activation-induced neuropathic pain in the sciatic nerve injury rat model

P2X₇ receptor is an important member of ATP-sensitive ionotropic P2X receptors family, which includes seven receptor subtypes (P2X₁-P2X₇). Recent evidence indicates that P2X₇R participates in the onset and persistence of neuropathic pain. In tetanic stimulation of the sciatic nerve model, P2X₇R was involved in the activation of microglia, but whether this happens in other neuropathic pain models remains unclear. In this study we used immunohistochemistry and Western blot to explore the relationship of P2X₇R expression with microglia activation, and with mechanical allodynia and thermal hypersensitivity in the chronic constriction of the sciatic nerve (CCI) rat model. The results show that following nerve ligature, mechanical allodynia and thermal hypersensitivity were developed within 3 days (d), peaked at 14 d and persisted for 21 d on the injured side. P2X₇R levels in the ipsilateral L4-6 spinal cord were increased markedly after injury and the highest levels were observed on day 14, significant difference was observed at I-IV layers of the dorsal horn. The change in P2X₇R levels in the spinal cord was consistent with the development of mechanical allodynia and thermal hypersensitivity. Intrathecal administration of the P2X₇R antagonist Brilliant Blue G (BBG) reversed CCI-induced mechanical allodynia and thermal hypersensitivity. Double-labeled immunofluorescence showed that P2X₇R expression were restricted to microglia, spinal microglia were activated after nerve injury, which was inhibited by BBG. These results indicated that spinal P2X₇R mediate microglia activation, this process may play an important role in development of mechanical allodynia and thermal hypersensitivity in CCI model.     

Fibronectin/integrin system is involved in P2X(4) receptor upregulation in the spinal cord and neuropathic pain after nerve injury

We have previously shown that activation of the ATP-gated ion channel subtype P2X(4) receptors (P2X(4)Rs) in the spinal cord, the expression of which is upregulated in microglia after nerve injury, is necessary for producing neuropathic pain. The upregulation of P2X(4)Rs in microglia is, therefore, a key process in neuropathic pain, but the mechanism remains unknown. Here, we find a fibronectin/integrin-dependent mechanism in the upregulation of P2X(4)Rs. Microglia cultured on dishes coated with fibronectin, an extracellular matrix molecule, expressed a higher level of P2X(4)R protein when compared with those cultured on control dishes. The increase was suppressed by echistatin, a peptide that selectively blocks beta(1) and beta(3)-containing integrins, and with a function-blocking antibody of beta(1) integrin. In in vivo studies, the upregulation of P2X(4)Rs in the spinal cord after spinal nerve injury was significantly suppressed by intrathecal administration of echistatin. Tactile allodynia in response to nerve injury and intrathecal administration of ATP- and fibronectin-stimulated microglia was inhibited by echistatin. Furthermore, intrathecal administration of fibronectin in normal rats increased the level of P2X(4)R protein in the spinal cord and produced tactile allodynia. Moreover, the fibronectin-induced allodynia was not observed in mice lacking P2X(4)R. Taken together with the results of our previous study showing an increase in the spinal fibronectin level after nerve injury, the present results suggest that the fibronectin/integrin system participates in the upregulation of P2X(4)R expression after nerve injury and subsequent neuropathic pain.     

P2X4 receptor channels form large noncytolytic pores in resting and activated microglia

P2X4 ATP-gated cation channels have been shown to contribute to the microglial component of central sensitization, making their functional regulation a key element in chronic pain pathologies. Here we show that prolonged activation of native P2X4 receptor channels by ATP induces opening of a pore permeable to NMDG(+) and large fluorescent dyes in BV-2 microglial cells and primary murine microglia. This intrinsic pore formation mechanism is potentiated by LPS treatment, known to upregulate P2X4 expression in microglial cells and to mimic the microglial activation observed in neuropathic pain states. Sustained activation of the P2X7 channel subtype, also expressed in microglia, induces a pore formation that requires pannexin hemichannels and leads to plasma membrane blebbing and cytotoxicity. In contrast, P2X4 pore formation is unaffected by the pannexin blocker carbenoxolone, does not induce cytoskeletal rearrangements and does not lead to cell death. Furthermore, we show that P2X4 pore dilation is modulated by phosphoinositides (PIP(n) ) levels as it is inhibited by wortmannin, a blocker of PIP(n) synthesis, suggesting possible regulation by phospholipase C-coupled pathways. Nonlethal P2X4 pore dilation could play a role in neuropathic pain by allowing the flux of large organic molecules in microglia. Different outcomes of P2X4 and P2X7 membrane permeabilization point to subtype-specific microglial responses to ATP in normal and pathological neuro-immune crosstalks.     

Role of hesperidin in P2X3 receptor-mediated neuropathic pain in the dorsal root ganglia

Aim: This study investigated whether the neuronal P2X3 receptor in rat dorsal root ganglia (DRG) mediated the effects of hesperidin on neuropathic pain.

Materials and methods: The chronic constriction injury (CCI) model was used as a model of neuropathic pain. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured. The mRNA and protein expression levels were assayed by real-time RT-PCR and Western blotting.

Results: The results showed that mechanical and thermal hyperalgesia in the CCI rats were increased as compared to those in the sham group. The expression levels of P2X3 mRNA and protein in CCI rats were higher than those in the sham group. Dual-labelling immunofluorescence showed that the elevated P2X3 receptor was co-expressed with the neuronal marker NeuN in the DRG of CCI rats. Hesperidin treatment decreased both the mechanical and thermal hyperalgesia, and upregulated P2X3 expression in the CCI rats. Hesperidin treatment also reduced the ERK1/2 phosphorylation in the DRG of CCI rats. Moreover, hesperidin inhibited the P2X3 agonist ATP-induced currents in HEK293 cells transfected with the P2X3 plasmid. Therefore, hesperidin treatment could reverse the elevated expression of neuronal P2X3 receptor and reduce the activation of ERK1/2 in the DRG of CCI rats.

Conclusions: Our findings suggested that hesperidin inhibited the nociceptive transmission mediated by the P2X3 receptor in neurons of DRG, and thus, relieved the mechanical and thermal hyperalgesia in CCI rats.     

Involvement of P2X4 and P2Y12 receptors in ATP-induced microglial chemotaxis

We previously reported that extracellular ATP induces membrane ruffling and chemotaxis of microglia and suggested that their induction is mediated by the Gi/o-protein coupled P2Y(12) receptor (P2Y(12)R). Here we report discovering that the P2X(4) receptor (P2X(4)R) is also involved in ATP-induced microglial chemotaxis. To understand the intracellular signaling pathway downstream of P2Y(12)R that underlies microglial chemotaxis, we examined the effect of two phosphatidylinositol 3'-kinase (PI3K) inhibitors, wortmannin, and LY294002, on chemotaxis in a Dunn chemotaxis chamber. The PI3K inhibitors significantly suppressed chemotaxis without affecting ATP-induced membrane ruffling. ATP stimulation increased Akt phosphorylation in the microglia, and the increase was reduced by the PI3K inhibitors and a P2Y(12)R antagonist. These results indicate that P2Y(12)R-mediated activation of the PI3K pathway is required for microglial chemotaxis in response to ATP. We also found that the Akt phosphorylation was reduced when extracellular calcium was chelated, suggesting that ionotropic P2X receptors are involved in microglial chemotaxis by affecting the PI3K pathway. We therefore tested the effect of various P2X(4)R antagonists on the chemotaxis, and the results showed that pharmacological blockade of P2X(4)R significantly inhibited it. Knockdown of the P2X(4) receptor in microglia by RNA interference through the lentivirus vector system also suppressed the microglial chemotaxis. These results indicate that P2X(4)R as well as P2Y(12)R is involved in ATP-induced microglial chemotaxis.     

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.     

Cold intolerance and neuropathic pain after peripheral nerve injury in upper extremity

Cold intolerance and pain can be a substantial problem in patients with peripheral nerve injury. We aimed at investigating the relationships among sensory recovery, cold intolerance, and neuropathic pain in patients affected by upper limb peripheral nerve injury (Sunderland type V) treated with microsurgical repair, followed by early sensory re‐education. In a cross‐sectional clinical study, 100 patients (male/female 81/19; age 40.5 ± 14.8 years and follow‐up 17 ± 5 months, mean ± SD), with microsurgical nerve repair and reconstruction in the upper extremity and subsequent early sensory re‐education, were evaluated, using Cold Intolerance Symptoms Severity questionnaire‐Italian version (CISS‐it, cut‐off pathology >30/100 points), CISS questionnaire‐12 item version (CISS‐12, 0‐46 points‐grouping: healthy that means no cold intolerance [0‐14], mild [15‐24], moderate [25‐34], severe [35‐42], very severe [43‐46] cold intolerance), probability of neuropathic pain (DouleurNeuropathique‐4; [DN4] 4/10), deep and superficial sensibility, tactile threshold (monofilaments), and two‐point discrimination (cutoff S2; Medical Research Council scale for sensory function; [MRC‐scale]). A high CISS score is associated with possible neuropathic pain (DN4 ≥ 4). Both a low CISS‐it score (ie, < 30) and DN4 < 4 is associated with good sensory recovery (MRC ≥ 2). In conclusion patients affected by upper limb peripheral nerve injuries with higher CISS scores more often suffer from cold intolerance and neuropathic pain, and the better their sensory recovery is, the less likely they are to suffer from cold intolerance and neuropathic pain.     

P2X4 receptors control the fate and survival of activated microglia

Microglia, the resident immune cells of the central nervous system, responds to brain disarrangements by becoming activated to contend with brain damage. Here we show that the expression of P2X4 receptors is upregulated in inflammatory foci and in activated microglia in the spinal cord of rats with experimental autoimmune encephalomyelitis (EAE) as well as in the optic nerve of multiple sclerosis patients. To study the role of P2X4 receptors in microgliosis, we activated microglia with LPS in vitro and in vivo. We observed that P2X4 receptor activity in vitro was increased in LPS‐activated microglia as assessed by patch‐clamp recordings. In addition, P2X4 receptor blockade significantly reduced microglial membrane ruffling, TNFα secretion and morphological changes, as well as LPS‐induced microglial cell death. Accordingly, neuroinflammation provoked by LPS injection in vivo induced a rapid microglial loss in the spinal cord that was totally prevented or potentiated by P2X4 receptor blockade or facilitation, respectively. Within the brain, microglia in the hippocampal dentate gyrus showed particular vulnerability to LPS‐induced neuroinflammation. Thus, microglia processes in this region retracted as early as 2 h after injection of LPS and died around 24 h later, two features which were prevented by blocking P2X4 receptors. Together, these data suggest that P2X4 receptors contribute to controlling the fate of activated microglia and its survival.GLIA 2014;62:171–184     

Therapeutic potential of progesterone in spinal cord injury-induced neuropathic pain: At the crossroads between neuroinflammation and N-methyl-D-aspartate receptor

In recent decades, an area of active research has supported the notion that progesterone promotes a wide range of remarkable protective actions in experimental models of nervous system trauma or disease, and has also provided a strong basis for considering this steroid as a promising molecule for modulating the complex maladaptive changes that lead to neuropathic pain, especially after spinal cord injury. In this review, we intend to give the readers a brief appraisal of the main mechanisms underlying the increased excitability of the spinal circuit in the pain pathway after trauma, with particular emphasis on those mediated by the activation of resident glial cells, the subsequent release of proinflammatory cytokines and their impact on N-methyl-D-aspartate receptor function. We then summarize the available preclinical data pointing to progesterone as a valuable repurposing molecule for blocking critical cellular and molecular events that occur in the dorsal horn of the injured spinal cord and are related to the development of chronic pain. Since the treatment and management of neuropathic pain after spinal injury remains challenging, the potential therapeutic value of progesterone opens new traslational perspectives to prevent central pain.     

Overexpression of P2X4 receptor in Schwann cells promotes motor and sensory functional recovery and remyelination via BDNF secretion after nerve injury

Of the seven P2X receptor subtypes, P2X4 receptor (P2X4R) is widely distributed in the central nervous system, including in neurons, astrocytes, and microglia. Accumulating evidence supports roles for P2X4R in the central nervous system, including regulating cell excitability, synaptic transmission, and neuropathic pain. However, little information is available about the distribution and function of P2X4R in the peripheral nervous system. In this study, we find that P2X4R is mainly localized in the lysosomes of Schwann cells in the peripheral nervous system. In cultured Schwann cells, TNF-a not only enhances the synthesis of P2X4R protein but also promotes P2X4R trafficking to the surface of Schwann cells. TNF-a-induced BDNF secretion in Schwann cells is P2X4R dependent. in vivo experiments reveal that expression of P2X4R in Schwann cells of injured nerves is strikingly upregulated following nerve crush injury. Moreover, overexpression of P2X4R in Schwann cells by genetic manipulation promotes motor and sensory functional recovery and accelerates nerve remyelination via BDNF release following nerve injury. Our results suggest that enhancement of P2X4R expression in Schwann cells after nerve injury may be an effective approach to facilitate the regrowth and remyelination of injured nerves.     

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.     

Potentiation of the P2X3 ATP receptor by PAR-2 in rat dorsal root ganglia neurons, through protein kinase-dependent mechanisms, contributes to inflammatory pain

Proinflammatory agents trypsin and mast cell tryptase cleave and activate protease-activated receptor-2 (PAR-2), which is expressed on sensory nerves and causes neurogenic inflammation. P2X3 is a subtype of the ionotropic receptors for adenosine 5'-triphosphate (ATP), and is mainly localized on nociceptors. Here, we show that a functional interaction of the PAR-2 and P2X3 in primary sensory neurons could contribute to inflammatory pain. PAR-2 activation increased the P2X3 currents evoked by α, β, methylene ATP in dorsal root ganglia (DRG) neurons. Application of inhibitors of either protein kinase C (PKC) or protein kinase A (PKA) suppressed this potentiation. Consistent with this, a PKC or PKA activator mimicked the PAR-2-mediated potentiation of P2X3 currents. In the in vitro phosphorylation experiments, application of a PAR-2 agonist failed to establish phosphorylation of the P2X3 either on the serine or the threonine site. In contrast, application of a PAR-2 agonist induced trafficking of the P2X3 from the cytoplasm to the plasma membrane. These findings indicate that PAR-2 agonists may potentiate the P2X3, and the mechanism of this potentiation is likely to be a result of translocation, but not phosphorylation. The functional interaction between P2X3 and PAR-2 was also confirmed by detection of the α, β, methylene-ATP-evoked extracellular signal-regulated kinases (ERK) activation, a marker of neuronal signal transduction in DRG neurons, and pain behavior. These results demonstrate a functional interaction of the protease signal with the ATP signal, and a novel mechanism through which protease released in response to tissue inflammation might trigger the sensation to pain through P2X3 activation.     

P2X7 receptor differentially modulates astroglial apoptosis and clasmatodendrosis in the rat brain following status epilepticus

Recently, it has been reported that astroglial loss/dysfunction plays a role in epileptogenesis. In addition, astroglial loss is accompanied by up‐regulation of P2X7 receptor expression in microglia. Therefore, we investigated whether P2X7 receptor is involved in astroglial damages induced by status epilepticus (SE). In the present study, astroglial loss showed the regional‐specific manner and the differential responses to P2X7 receptor functions. Both OxATP and brilliant blue G (P2X7 receptor antagonists) infusion prevented apoptotic astroglial loss in the molecular layer of the dentate gyrus and the frontoparietal cortex, while it promoted clasmatodendrosis in the CA1 region as compared to saline treatment. In contrast, BzATP (a P2X7 receptor agonist) treatment exacerbated apoptotic astroglial loss in the molecular layer of the dentate gyrus and the frontoparietal cortex, but alleviated SE‐induced astroglial swelling in the CA1 region. Astroglial loss in the piriform cortex was not affected by P2X7 receptor agonist‐ or antagonist‐infusion. These findings suggest that P2X7 receptor function differently modulates SE‐induced astroglial loss in distinct brain regions. © 2010 Wiley Periodicals, Inc.