Purinergic P2X7 Receptor Research Articles

Mitochondrial Localization and Function of the Purinergic Receptor P2X7.

Mitochondrial Localization and Function of the Purinergic Receptor P2X7.

Catestatin enhances ATP-induced activation of glial cells mediated by purinergic receptor P2X4

The activation of glial cells and its possible mechanism play an extremely important role in understanding the pathophysiological process of some clinical diseases, and catestatin (CST) is involved in regulating this activation. In this project, we found that CST could enhance the activation of satellite glial cells (SGCs) and microglial cells and that the expression of P2X4 was increased; the co-expression of the P2X4 receptor with glial fibrillary acidic protein (GFAP) and the P2X4 receptor with CD11b was also increased significantly in glial cells of the ATP + CST group, and TNF-α and IL-1β also showed a rising trend; the expression of phosphorylated ERK1/2 was also increased in the ATP + CST group. In summary, we conclude that CST could enhance ATP-induced activation of SGCs and microglial cells mediated by the P2X4 receptor and that the ERK1/2 signaling pathway may be involved in this activation process.

Restraint of Human Skin Fibroblast Motility, Migration, and Cell Surface Actin Dynamics, by Pannexin 1 and P2X7 Receptor Signaling.

Wound healing is a dynamic process required to maintain skin integrity and which relies on the precise migration of different cell types. A key molecule that regulates this process is ATP. However, the mechanisms involved in extracellular ATP management are poorly understood, particularly in the human dermis. Here, we explore the role, in human fibroblast migration during wound healing, of Pannexin 1 channels and their relationship with purinergic signals and in vivo cell surface filamentous actin dynamics. Using siRNA against Panx isoforms and different Panx1 channel inhibitors, we demonstrate in cultured human dermal fibroblasts that the absence or inhibition of Panx1 channels accelerates cell migration, increases single-cell motility, and promotes actin redistribution. These changes occur through a mechanism that involves the release of ATP to the extracellular space through a Panx1-dependent mechanism and the activation of the purinergic receptor P2X7. Together, these findings point to a pivotal role of Panx1 channels in skin fibroblast migration and suggest that these channels could be a useful pharmacological target to promote damaged skin healing.

Intracolonic Mustard Oil Induces Visceral Pain in Mice by TRPA1-Dependent and -Independent Mechanisms: Role of Tissue Injury and P2X Receptors.

Both TRPA1 and purinergic P2X receptors have been proposed as potential targets for the treatment of visceral pain. We found that the intracolonic administration of a low dose mustard oil (0.5%), a well-known TRPA1 agonist, produced nociceptive responses and abdominal wall referred mechanical hyperalgesia, without inducing apparent tissue damage. Both nociceptive responses and referred hyperalgesia were abolished by the ablation of TRPV1-expressing neurons (and the consequent ablation of TRPA1+ nociceptors) by resiniferatoxin (RTX) treatment, and by the TRPA1 antagonist AP18. However, a higher dose of mustard oil (2.5%) damaged the colonic epithelium and induced pERK activation in the spinal cord, and these processes were clearly independent of TRPV1-expressing neurons ablated by RTX. This higher dose of mustard oil induced nociceptive responses and referred mechanical hyperalgesia which were insensitive or only slightly sensitive to resiniferatoxin or AP18, but were markedly reduced by the P2X antagonist TNP-ATP, which is known to inhibit nociceptive actions induced by ATP released from injured tissues. In conclusion, whereas a low dose of intracolonic mustard oil induces visceral pain in a manner fully dependent on TRPA1 actions, when a high dose of this chemical irritant is used, visceral pain becomes mostly independent of TRPA1 activation but clearly enhanced by ATP purportedly released by the damaged colonic epithelium. Therefore, TRPA1 inhibition is not sufficient to substantially decrease visceral pain during tissue injury, whereas purinergic antagonism appears to be a more effective strategy.

The effects of simulated childbirth trauma on the gene expression of neurotransmitter receptors in the bladder of female rats

To investigate the effects of simulated childbirth on the gene expression of parasympathetic muscarinic, purinergic (P2X), and neurokinin receptors of lower urinary tract in rats. In all, twenty-four primiparous pregnant Sprague-Dawley female rats were equally divided into three groups: (1). Control group; 8 rats, (2) intra-vaginal balloon dilation for 2 h group; 8 rats, (3) and for 4 h group; 8 rats. After balloon dilatation for 4 months, all rats were sacrificed. We analyzed the gene expression of parasympathetic muscarinic, purinergic (P2X), and neurokinin receptors by real-time quantitative PCR (q-PCR). We quantified pro-inflammatory cytokines of TNF-α and IL-6 by Enzyme-linked immunosorbent assays (ELISA). The urodynamic parameters and micturition frequency by cystometry (CMG) were recorded. Our results showed that the balloon dilation significantly increased micturition frequency and modified peak micturition pressure compare to those in the control groups. Balloon dilation significantly decreased voiding interval and bladder volume compared to those in the control groups. Gene expressions of M3 muscarinic, P2X3 purinergic receptors, and significantly increased following balloon dilation for 2 hours and 4 hours than those in the control group. In addition, we found that NK1R and NK3R receptors were significantly decreased after balloon dilation compare to control group. The marked increase of TNF-α and IL-6 were also seen in the 2 balloon groups. The results of our study suggested that birth trauma may impair the function of urinary tract, this being partly related to the changes in the gene expression of the neurotransmitter receptors of the lower urinary tract.

Astragalin Alleviates Neuropathic Pain by Suppressing P2X4-Mediated Signaling in the Dorsal Root Ganglia of Rats.

Neurologic damage often leads to neuropathic pain, for which there are no effective treatments owing to its complex pathogenesis. The purinergic receptor P2X4 is closely associated with neuropathic pain. Astragalin (AST), a compound that is used in traditional Chinese medicine, has protective effects against allergic dermatitis and neuronal injury, but its mechanism of action is not well understood. The present study investigated whether AST can alleviate neuropathic pain in a rat model established by chronic constriction injury (CCI) to the sciatic nerve. The model rats exhibited pain behavior and showed increased expression of P2X4 and the activated satellite glial cell (SGC) marker glial fibrillary acidic protein in dorsal root ganglia (DRG). AST treatment partly abrogated the upregulation of P2X4, inhibited SGC activation, and alleviated pain behavior in CCI rats; it also suppressed ATP-activated currents in HEK293 cells overexpressing P2X4. These data demonstrate that AST relieves neuropathic pain by inhibiting P2X4 and SGC activation in DRG, highlighting its therapeutic potential for clinical pain management.

Effect of P2X purinergic receptors in tumor progression and as a potential target for anti-tumor therapy.

The development of tumors is a complex pathological process involving multiple factors, multiple steps, and multiple genes. Their prevention and treatment have always been a difficult problem at present. A large number of studies have proved that the tumor microenvironment plays an important role in the progression of tumors. The tumor microenvironment is the place where tumor cells depend for survival, and it plays an important role in regulating the growth, proliferation, apoptosis, migration, and invasion of tumor cells. P2X purinergic receptors, which depend on the ATP ion channel, can be activated by ATP in the tumor microenvironment, and by mediating tumor cells and related cells (such as immune cells) in the tumor microenvironment. They play an important regulatory role on the effects of the skeleton, membrane fluidity, and intracellular molecular metabolism of tumor cells. Therefore, here, we outlined the biological characteristics of P2X purinergic receptors, described the effect of tumor microenvironment on tumor progression, and discussed the effect of ATP on tumor. Moreover, we explored the role of P2X purinergic receptors in the development of tumors and anti-tumor therapy. These data indicate that P2X purinergic receptors may be used as another potential pharmacological target for tumor prevention and treatment.

Effect of the conditioned medium of mesenchymal stem cells on the expression levels of P2X4 and P2X7 purinergic receptors in the spinal cord of rats with neuropathic pain.

Recent studies have shown that mesenchymal stem cells (MSCs) and their conditioned medium (CM) have potential therapeutic effects in animal models of neuropathic pain (NP). However, the mechanisms underlying these effects are not fully understood. Because of the leading involvement of purinergic receptors in the pathogenesis of NP, this study aimed to investigate the effect of MSCs-CM on the expression levels of P2X4 and P2X7 receptors in a rat model of NP induced by chronic constriction injury (CCI) of the sciatic nerve. CM was prepared from the rats' bone marrow-derived MSCs culture. After that, NP rats were treated by intraperitoneal injection of CM, or Dulbecco's modified Eagle's medium (DMEM) 1day before and 7 and 11days after CCI surgery. The NP status was assessed in the treated animals using behavioral tests, including mechanical allodynia and thermal hyperalgesia, on days - 1, 3, 6, 9, 12, and 15 of the study. At the end of the study (Day 15), the animals were sacrificed, and the relative gene expression of P2X4 and P2X7 receptors were measured in the spinal cord using quantitative real-time PCR. The results demonstrated that in the CM-treated NP rats, mechanical allodynia and thermal hyperalgesia were significantly reduced compared with the DMEM-treated group. In addition, the expression levels of P2X4 and P2X7 receptors were noticeably prevented in the CM-treated group than the control group. These findings indicate that the antinociceptive effects of CM in the NP rats are partly mediated through preventing the upregulation of P2X4 and P2X7 receptors in the spinal cord.

PRG-1 relieves pain and depressive-like behaviors in rats of bone cancer pain by regulation of dendritic spine in hippocampus.

Rationale: Pain and depression, which tend to occur simultaneously and share some common neural circuits and neurotransmitters, are highly prevalent complication in patients with advanced cancer. Exploring the underlying mechanisms is the cornerstone to prevent the comorbidity of chronic pain and depression in cancer patients. Plasticity-related gene 1 (PRG-1) protein regulates synaptic plasticity and brain functional reorganization during neuronal development or after cerebral lesion. Purinergic P2X7 receptor has been proposed as a therapeutic target for various pain and neurological disorders like depression in rodents. In this study, we investigated the roles of PRG-1 in the hippocampus in the comorbidity of pain and depressive-like behaviors in rats with bone cancer pain (BCP).Methods: The bone cancer pain rat model was established by intra-tibial cell inoculation of SHZ-88 mammary gland carcinoma cells. The animal pain behaviors were assessed by measuring the thermal withdrawal latency values by using radiant heat stimulation and mechanical withdrawal threshold by using electronic von Frey anesthesiometer, and depressive-like behavior was assessed by sucrose preference test and forced swim test. Alterations in the expression levels of PRG-1 and P2X7 receptor in hippocampus were separately detected by using western blot, immunofluorescence and immunohistochemistry analysis. The effects of intra-hippocampal injection of FTY720 (a PRG-1/PP2A interaction activator), PRG-1 overexpression or intra-hippocampal injection of A438079 (a selective competitive P2X7 receptor antagonist) were also observed.Results: Carcinoma intra-tibia injection caused thermal hyperalgesia, mechanical allodynia and depressive-like behaviors in rats, and also induced the deactivation of neurons and dendritic spine structural anomalies in the hippocampus. Western blot, immunofluorescence and immunohistochemistry analysis showed an increased expression of PRG-1 and P2X7 receptor in the hippocampus of BCP rats. Intra-hippocampal injection of FTY720 or A438079 attenuated both pain and depressive-like behaviors. Furthermore, overexpression of PRG-1 in hippocampus has similar analgesic efficacy to FTY720. In addition, they rescued neuron deactivation and dendritic spine anomalies.Conclusion: The results suggest that both PRG-1 and P2X7 receptor in the hippocampus play important roles in the development of pain and depressive-like behaviors in bone cancer condition in rats by dendritic spine regulation via P2X7R/PRG-1/PP2A pathway.

P2X4 receptor participates in autophagy regulation in Parkinson's disease.

Dysfunctional autophagy often occurs during the development of neurodegenerative diseases, such as Parkinson's disease, Huntington's disease, and Alzheimer's disease. The purinergic P2X4 receptor is an ATP-gated ion channel that is widely expressed in the microglia, astrocytes, and neurons of the central and peripheral nervous systems. P2X4R is involved in the regulation of cellular excitability, synaptic transmission, and neuroinflammation. However, the role played by P2X4R in Parkinson's disease remains poorly understood. Rat models of Parkinson's disease were established by injecting 6-hydroxydopamine into the substantia nigra pars compacta. P2X4R-targeted small interfering RNA (siRNA) was injected into the same area 1 week before injury induction to inhibit the expression of the P2X4 receptor. The results showed that the inhibition of P2X4 receptor expression in Parkinson's disease model rats reduced the rotation behavior induced by apomorphine treatment, increased the latency on the rotarod test, and upregulated the expression of tyrosine hydroxylase, brain-derived neurotrophic factor, LC3-II/LC3-I, Beclin-1, and phosphorylated tropomyosin receptor kinase B (TrkB) in brain tissue, while simultaneously reducing p62 levels. These findings suggest that P2X4 receptor activation might inhibit neuronal autophagy through the regulation of the brain-derived neurotrophic factor/TrkB signaling pathway, leading to dopaminergic neuron damage in the substantia nigra and the further inhibition of P2X4 receptor-mediated autophagy. These results indicate that P2X4 receptor might serve as a potential novel target for the treatment of Parkinson's disease. This study was approved by the Animal Ethics Committee of Affiliated Hospital of Qingdao University (approval No. QYFYWZLL26119) on April 12, 2016.

The interaction between P2X3 and TRPV1 in the dorsal root ganglia of adult rats with different pathological pains

Peripheral inflammatory and neuropathic pain are closely related to the activation of purinergic receptor P2X ligand-gated ion channel 3 (P2X3) and transient receptor potential vanilloid 1 (TRPV1), but the interaction between P2X3 and TRPV1 in different types of pathological pain has rarely been reported. In this study, complete Freund’s adjuvant (CFA)-induced inflammatory pain and spared nerve injury (SNI)-induced neuropathic pain models were established in adult rats. The interactions between P2X3 and TRPV1 in the dorsal root ganglion were observed by pharmacological, co-immunoprecipitation, immunofluorescence and whole-cell patch-clamp recording assays. TRPV1 was shown to promote the induction of spontaneous pain caused by P2X3 in the SNI model, but the induction of spontaneous pain behaviour by TRPV1 was not completely dependent on P2X3 in vivo. In both the CFA and SNI models, the activation of peripheral P2X3 enhanced the effect of TRPV1 on spontaneous pain, while the inhibition of peripheral TRPV1 reduced the induction of spontaneous pain by P2X3 in the CFA model. TRPV1 and P2X3 had inhibitory effects on each other in the inflammatory pain model. During neuropathic pain, P2X3 facilitated the function of TRPV1, while TRPV1 had an inhibitory effect on P2X3. These results suggest that the mutual effects of P2X3 and TRPV1 differ in cases of inflammatory and neuropathic pain in rats.

Flow cytometry: An accurate tool for screening P2RX7 modulators

AbstractThe P2X purinergic receptor 7 (P2RX7) is a poorly selective ATP‐gated ion channel. Although P2RX7 binds ATP with relatively low affinity, prolonged activation can lead to nonselective membrane pore formation. Indeed, brief exposure to ATP triggers a rapid Ca2+ influx, whereas prolonged exposure to high ATP concentrations results in the passage of larger organic molecules. P2RX7 is involved in the physiopathology of a number of diseases and has notably emerged as a potential therapeutic target in inflammation, neuropathic pain, Alzheimer's disease, and cancer—prompting growing interest in the synthesis of novel P2RX7 modulators and the development of reliable, stringent screening methods. In the present study, we developed methods based on conventional flow cytometry, imaging flow cytometry and spectral flow cytometry and used them to measure P2RX7's activity upon activation by 3'‐O‐(4‐benzoyl)benzoyl ATP. We also demonstrated the use of the highly sensitive DNA‐intercalating dye TO‐PRO‐3 to determine P2RX7's large pore activity. The simultaneous quantification of calcium influx (Fluo‐3 AM), large pore opening (TO‐PRO‐3), and viability (propidium iodide) is a very efficient method for low‐ to medium‐throughput screening of P2RX7 modulators. Agonist and antagonist potencies can be accurately evaluated. Spectral cytometry notably enabled us to assay several biological activities while correcting for the intrinsic fluorescence of the screened compounds—otherwise a well‐known limitation of fluorescence‐based screening. Hence, spectral cytometry appears to be a useful, novel tool for drug candidate screening.

Performance of P2x7 and P2Y Purinergic Receptors as an Inhibiting Factor in the Progression of Pulmonary Neoplastic Cells

Different pathological processes are considered in lung cancer, such as nicotine deposition, oxidative stress, deregulation of metal ions and chronic inflammation. Purine signaling is involved in all processes, suggesting the importance of nucleotide receptors (P2X7 and P2Y) and adenosine receptors present in lung cells. The accumulation of toxic substances promotes chronic inflammation and cellular alteration, which induces the release of Adenosine Triphosphate (ATP) in the extracellular space, thus stimulating P2X7 receptors. The activation of P2X7 promotes an increase in the synthesis and release of some pro-inflammatory mediators. P2Y works by promoting the increase of intracellular calcium levels and, P2X7, changing cell permeability. Thus, drugs can be developed with a specific targeting for such recipients, thus provid additional therapeutic options to those existing.

The expression of P2X7 receptor in human induced pluripotent stem cell‐derived cellular model of Alzheimer’s disease

AbstractBackgroundWe aim to elucidate the role of the purinergic receptor P2X7 in the development and progression of the Alzheimer’s disease (AD) pathology in human induced pluripotent stem cell‐derived cellular model. Our model consists of iPSC‐derived mixed neuronal and astrocytic cells, together with human iPSC‐derived microglia‐like cells. Since jointly with amyloid‐β and tau pathology, neuroinflammation presents one of the main factors in AD. Thus, having microglia in the cellular model of AD is unavoidable.MethodiPSC‐derived neuronal cultures were generated using the dual SMAD inhibition protocol, while microglial cells were generated through a 3D differentiation protocol, using bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), and stem cell factor (SCF). Further differentiation was forced by adding macrophage colony‐stimulating factor (M‐CSF) and interleukin‐3 (IL‐3). Expression of cellular markers, as well as the studied P2X7R, was verified via ELISA, immunocytochemistry, western blot and RT‐qPCR.ResultOur neuronal cell cultures obtained from AD patients have been previously reported to show major pathologic signs of AD, thus, suitable for AD modelling. The acquired microglia‐like cells expressed Iba1, CX3CR1, CD11c, and TMEM119, which confirms their suitability for further use. Both neuronal and microglial cells express the investigated P2X7 receptor. Moreover, based on our preliminary results, the expression of P2X7R in neurons seems to be diminished when co‐cultivated with microglia. This might suggest a temporary modulatory mechanism, activated to supplement the function of the receptor in the absence of microglia.ConclusionBased on our results, the presented cellular system offers an excellent model that can be used for studying the molecular basis of age‐related neurodegenerative diseases such as Alzheimer’s disease. Future experiments will explore the effects of P2X7R antagonism on AD pathology in our cellular model. P2X7R seems to be actively engaged in AD pathological processes, and thus, the modulation of the receptor’s activity with antagonists might present a good therapeutic approach for AD. Therefore, this research helps to validate the correlation between animal and human studies of P2X7R; and will be extended to the evaluation of the therapeutic potential of selected P2X7R antagonists in Alzheimer’s disease.

Chemoresistance in Breast Cancer Patients Associated With Changes in P2X7 and A2A Purinergic Receptors in CD8+ T Lymphocytes.

Breast cancer (BRCA) is the most frequent cancer type that afflicts women. Unfortunately, despite all the current therapeutic strategies, many patients develop chemoresistance hampering the efficacy of treatment. Hence, an early indicator of therapy efficacy might aid in the search for better treatment and patient survival. Although emerging evidence indicates a key role of the purinergic receptors P2X7 and A2A in cancer, less is known about their involvement in BRCA chemoresistance. In this sense, as the chemotherapeutic treatment stimulates immune system response, we evaluated the expression and function of P2X7 and A2A receptors in CD8+ T cells before and four months after BRCA patients received neoadjuvant chemotherapy. The results showed an increase in the levels of expression of P2X7 and a decrease in the expression of A2A in CD8+ T cells in non-chemoresistant (N-CHR) patients, compared to chemoresistant (CHR) patients. Interestingly, in CHR patients, reduced expression of P2X7 occurs along with a decrease in the CD62L shedding and the production of IFN-γ. In the case of the A2A function, the inhibition of IFN-γ production was not observed after chemotherapy in CHR patients. A possible relationship between the modulation of the expression and function of the P2X7 and A2A receptors was found, according to the molecular subtypes, where the patients that were triple-negative and human epidermal growth factor receptor 2 (HER2)-enriched presented more alterations. Comorbidities such as overweight/obesity and type 2 diabetes mellitus (T2DM) participate in the abnormalities detected. Our results demonstrate the importance of purinergic signaling in CD8+ T cells during chemoresistance, and it could be considered to implement personalized therapeutic strategies.

New Horizons: Does Mineralocorticoid Receptor Activation by Cortisol Cause ATP Release and COVID-19 Complications?

This paper attempts to explain how the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus causes the complications that make coronavirus disease 2019 (COVID-19) a serious disease in specific patient subgroups. It suggests that cortisol-associated activation of the mineralocorticoid receptor (MR) in epithelial and endothelial cells infected with the virus stimulates the release of adenosine 5′-triphosphate (ATP), which then acts back on purinergic receptors. In the lung this could produce the nonproductive cough via purinergic P2X3 receptors on vagal afferent nerves. In endothelial cells it could stimulate exocytosis of Weibel-Palade bodies (WPBs) that contain angiopoietin-2, which is important in the pathogenesis of acute respiratory distress syndrome (ARDS) by increasing capillary permeability and von Willebrand factor (VWF), which mediates platelet adhesion to the endothelium and hence clotting. Angiopoietin-2 and VWF levels both are markedly elevated in COVID-19–associated ARDS. This paper offers an explanation for the sex differences in SARS-CoV-2 complications and also for why these are strongly associated with age, race, diabetes, and body mass index. It also explains why individuals with blood group A have a higher risk of severe infection than those with blood group O. Dexamethasone has been shown to be of benefit in coronavirus ARDS patients and has been thought to act as an anti-inflammatory drug. This paper suggests that a major part of its effect may be due to suppression of cortisol secretion. There is an urgent need to trial the combination of dexamethasone and an MR antagonist such as spironolactone to more effectively block the MR and hence the exocytosis of WPBs.

Metformin alleviates hydrogen peroxide-induced inflammation and oxidative stress via inhibiting P2X7R signaling in spinal cord tissue cells neurons.

Metformin, the first medication that is often prescribed for the treatment of type 2 diabetes mellitus, was recently found to be neuroprotective. To study the mechanism underlying the neuroprotective effect of metformin, we pretreated primary spinal cord neurons with 50 µM or 100 µM metformin for 2 h prior to treatment with hydrogen peroxide (H2O2) for up to 48 h. Our results showed that H2O2 increased the expression of purinergic receptor P2X7 (P2X7R) in spinal cord neurons, which promoted the downstream pro-inflammatory cytokines release and oxidative stress. We found that metformin could reverse these pro-inflammatory and pro-oxidative effects of H2O2. Besides, P2X7R knockdown by siRNA suppressed H2O2-induced pro-inflammatory cytokine release and oxidative stress response. In conclusion, our results show that metformin can alleviate H2O2-induced inflammation and oxidative stress via modulating the P2X7R signaling pathway.

Dissection of P2X4 and P2X7 Receptor Current Components in BV-2 Microglia.

Microglia cells represent the immune system of the central nervous system. They become activated by ATP released from damaged and inflamed tissue via purinergic receptors. Ionotropic purinergic P2X4 and P2X7 receptors have been shown to be involved in neurological inflammation and pain sensation. Whether the two receptors assemble exclusively as homotrimers or also as heterotrimers is still a matter of debate. We investigated the expression of P2X receptors in BV-2 microglia cells applying the whole-cell voltage-clamp technique. We dissected P2X4 and P2X7 receptor-mediated current components by using specific P2X4 and P2X7 receptor blockers and by their characteristic current kinetics. We found that P2X4 and P2X7 receptors are activated independently from each other, indicating that P2X4/P2X7 heteromers are not of functional significance in these cells. The pro-inflammatory mediators lipopolysaccharide and interferon γ, if applied in combination, upregulated P2X4, but not P2X7 receptor-dependent current components also arguing against phenotypically relevant heteromerization of P2X4 and P2X7 receptor subunits.

P2X7 Receptor-Dependent Layer-Specific Changes in Neuron-Microglia Reactivity in the Prefrontal Cortex of a Phencyclidine Induced Mouse Model of Schizophrenia.

Background: It has been consistently reported that the deficiency of the adenosine triphosphate (ATP) sensitive purinergic receptor P2X7 (P2X7R) ameliorates symptoms in animal models of brain diseases.Objective: This study aimed to investigate the role of P2X7R in rodent models of acute and subchronic schizophrenia based on phencyclidine (PCP) delivery in animals lacking or overexpressing P2X7R, and to identify the underlying mechanisms involved.Methods: The psychotomimetic effects of acute i.p. PCP administration in C57Bl/6J wild-type, P2X7R knockout (P2rx7−/−) and overexpressing (P2X7-EGFP) young adult mice were quantified. The medial prefrontal cortex (mPFC) of P2rx7−/− and heterozygous P2X7-EGFP acutely treated animals was characterized through immunohistochemical staining. The prefrontal cortices of young adult P2rx7−/− and P2rx7tg/+ mice were examined with tritiated dopamine release experiments and the functional properties of the mPFC pyramidal neurons in layer V from P2rx7−/− mice were assessed by patch-clamp recordings. P2rx7−/− animals were subjected to a 7 days subchronic systemic PCP treatment. The animals working memory performance and PFC cytokine levels were assessed.Results: Our data strengthen the hypothesis that P2X7R modulates schizophrenia-like positive and cognitive symptoms in NMDA receptor antagonist models in a receptor expression level-dependent manner. P2X7R expression leads to higher medial PFC susceptibility to PCP-induced circuit hyperactivity. The mPFC of P2X7R knockout animals displayed distinct alterations in the neuronal activation pattern, microglial organization, specifically around hyperactive neurons, and were associated with lower intrinsic excitability of mPFC neurons.Conclusions: P2X7R expression exacerbated PCP-related effects in C57Bl/6J mice. Our findings suggest a pleiotropic role of P2X7R in the mPFC, consistent with the observed behavioral phenotype, regulating basal dopamine concentration, layer-specific neuronal activation, intrinsic excitability of neurons in the mPFC, and the interaction of microglia with hyperactive neurons. Direct measurements of P2X7R activity concerning microglial ramifications and dynamics could help to further elucidate the molecular mechanisms involved.

Assessment of neuroinflammation in the aging hippocampus using large-molecule microdialysis: Sex differences and role of purinergic receptors

Aging is associated with an enhanced neuroinflammatory response to acute immune challenge, often termed “inflammaging.” However, there are conflicting reports about whether baseline levels of inflammatory markers are elevated under ambient conditions in the aging brain, or whether such changes are observed predominantly in response to acute challenge. The present studies utilized two distinct approaches to assess inflammatory markers in young and aging Fischer 344 rats. Experiment 1 examined total tissue content of inflammatory markers from hippocampus of adult (3 month), middle-aged (12 month), and aging (18 month) male Fischer (F) 344 rats using multiplex analysis (23-plex). Though trends emerged for several cytokines, no significant differences in basal tissue content were observed across the 3 ages examined. Experiment 2 measured extracellular concentrations of inflammatory factors in the hippocampus from adult (3 month) and aging (18 month) males and females using large-molecule in vivo microdialysis. Although few significant aging-related changes were observed, robust sex differences were observed in extracellular concentrations of CCL3, CCL20, and IL-1α. Experiment 2 also evaluated the involvement of the P2X7 purinergic receptor in neuroinflammation using reverse dialysis of the selective agonist BzATP. BzATP produced an increase in IL-1α and IL-1β release and rapidly suppressed the release of CXCL1, CCL2, CCL3, CCL20, and IL-6. Other noteworthy sex by aging trends were observed in CCL3, IL-1β, and IL-6. Together, these findings provide important new insight into late-aging and sex differences in neuroinflammation, and their regulation by the P2X7 receptor.