Central P2Y12 receptor blockade alleviates inflammatory and neuropathic pain and cytokine production in rodents.

In this study the role of P2Y12 receptors (P2Y12R) was explored in rodent models of inflammatory and neuropathic pain and in acute thermal nociception. In correlation with their activity to block the recombinant human P2Y12R, the majority of P2Y12R antagonists alleviated mechanical hyperalgesia dose-dependently, following intraplantar CFA injection, and after partial ligation of the sciatic nerve in rats. They also caused an increase in thermal nociceptive threshold in the hot plate test. Among the six P2Y12R antagonists evaluated in the pain studies, the selective P2Y12 receptor antagonist PSB-0739 was most potent upon intrathecal application. P2Y12R mRNA and IL-1ß protein were time-dependently overexpressed in the rat hind paw and lumbar spinal cord following intraplantar CFA injection. This was accompanied by the upregulation of TNF-α, IL-6 and IL-10 in the hind paw. PSB-0739 (0.3mg/kg i.t.) attenuated CFA-induced expression of cytokines in the hind paw and of IL-1ß in the spinal cord. Subdiaphragmatic vagotomy and the α7 nicotinic acetylcholine receptor antagonist MLA occluded the effect of PSB-0739 (i.t.) on pain behavior and peripheral cytokine induction. Denervation of sympathetic nerves by 6-OHDA pretreatment did not affect the action of PSB-0739. PSB-0739, in an analgesic dose, did not influence motor coordination and platelet aggregation. Genetic deletion of the P2Y12R in mice reproduced the effect of P2Y12R antagonists on mechanical hyperalgesia in inflammatory and neuropathic pain models, on acute thermal nociception and on the induction of spinal IL-1ß. Here we report the robust involvement of the P2Y12R in inflammatory pain. The anti-hyperalgesic effect of P2Y12R antagonism could be mediated by the inhibition of both central and peripheral cytokine production and involves α7-receptor mediated efferent pathways.

The absence of P2X7 receptors (P2rx7) on non-haematopoietic cells leads to selective alteration in mood-related behaviour with dysregulated gene expression and stress reactivity in mice.

The purpose of this study was to explore how genetic deletion and pharmacological antagonism of the P2X7 receptor (P2rx7) alter mood-related behaviour, gene expression and stress reactivity in the brain. The forced swim test (FST), tail suspension test (TST) and amphetamine-induced hyperlocomotion (AH) tests were used in wild-type (P2rx7(+/+)) and P2rx7-deficient (P2rx7(-/-)) mice. Biogenic amine levels were analysed in the amygdala and striatum, adrenocorticotropic hormone (ACTH) and corticosterone levels were measured in the plasma and pituitary after restraint stress. Chimeric mice were generated by bone marrow transplantation. A whole genome microarray analysis with real-time polymerase chain reaction validation was performed on the amygdala. In the absence of P2rx7s decreased behavioural despair in the FST, reduced immobility in the TST and attenuated amphetamine-induced hyperactivity were detected. Basal norepinephrine levels were elevated in the amygdala, whereas stress-induced ACTH and corticosterone responses were alleviated in P2rx7(-/-) mice. Sub-acute treatment with the selective P2rx7 antagonist, Brilliant Blue G, reproduced the effect of genetic deletion in the TST and AH test in P2rx7(+/+) but not P2rx7(-/-) mice. No change in behavioural phenotype was observed in chimeras lacking the P2rx7 in their haematopoietic compartment. Whole genome microarray analysis indicated a widespread up- and down-regulation of genes crucial for synaptic function and neuroplasticity by genetic deletion. Here, we present evidence that the absence of P2rx7s on non-haematopoietic cells leads to a mood-stabilizing phenotype in several behavioural models and suggest a therapeutic potential of P2rx7 antagonists for the treatment of mood disorders.

The role of purinergic signaling in depressive disorders.

The purinergic signaling system consists of transporters, enzymes and receptors responsible for the synthesis, release, action and extracellular inactivation of adenosine 5'-triphosphate (ATP) and its extracellular breakdown product adenosine. The actions of ATP are mediated ionotropic P2X and metabotropic P2Y receptor subfamilies, whilst the actions of adenosine are mediated by P1 adenosine receptors. Purinergic signaling pathways are widely expressed in the central nervous system (CNS) and participate in its normal and pathological functions. Among P2X receptors, the P2X7 receptor (P2rx7) has received considerable interest in both basic and clinical neuropsychiatric research because of its profound effects in animal CNS pathology and its potential involvement as a susceptibility gene in mood disorders. Although genetic findings were not always consistently replicated, several studies demonstrated that single nucleotide polymorphisms (SNPs) in the human P2X7 gene (P2RX7) show significant association with major depressive disorder and bipolar disorder. Animal studies revealed that the genetic knock-down or pharmacological antagonism leads to reduced depressive-like behavior, attenuated response in mania-model and alterations in stress reactivity. A potential mechanism of P2rx7 activation on mood related behavior is increased glutamate release, activation of extrasynaptic NMDA receptors and subsequent enduring changes in neuroplasticity. In addition, dysregulation of monoaminergic transmission and HPA axis reactivity could also contribute to the observed changes in behavior. Besides P2rx7, the inhibition of adenosine A1 and A2A receptors also mediate antidepressant-like effects in animal experiments. In conclusion, despite contradictions between existing data, these findings point to the therapeutic potential of the purinergic signaling system in mood disorders.

Endocannabinergic modulation of interleukin-1ß in mouse hippocampus under basal conditions and after in vivo systemic lipopolysaccharide stimulation.

OBJECTIVE: Cannabinoids play an important role in the suppression of proinflammatory cytokine production in the periphery and brain. In this study, we explored whether endogenous activation of cannabinoid (CB) 1 receptors (CB1Rs) affects interleukin (IL)-1ß levels in the mouse hippocampus under basal conditions and following stimulation with in vivo bacterial lipopolysaccharide (LPS, 250 µg/kg i.p.). METHODS: IL-1ß levels were determined in the hippocampi of wild-type (WT), CB1R-/- and P2X7 receptor (P2X7R)-/- mice using an ELISA kit. RESULTS: Basal but not LPS-induced IL-1ß levels were downregulated when CB1R function was abrogated by genetic deletion, suggesting that endocannabinoids contributed to basal IL-1ß content in the mouse hippocampus. AM251 (3 mg/kg i.p.), an antagonist of CB1Rs, also inhibited basal IL-1ß protein in WT but not in CB1R-/- mice. In the absence of P2X7R, LPS-induced IL-1ß production was lower, while the inhibitory effect of CB1R antagonists on basal IL-1ß was significantly attenuated. The LPS-induced elevation in IL-1ß production was decreased in the presence of AM251 and AM281, with no significant difference between WT and P2X7R-/- mice. CONCLUSIONS: CB1Rs are responsible for the modulation of basal IL-1ß levels in the hippocampus, while the effects of CB1 antagonists on systemic LPS-induced IL-1ß concentrations are independent of CB1Rs.

Disrupted Social Hierarchy in Prenatally Valproate-Exposed Autistic-Like Rats.

Autism spectrum disorder (ASD) is characterized by impaired socio-communicational function, repetitive and restricted behaviors. Valproic acid (VPA) was reported to increase the prevalence of ASD in humans as a consequence of its use during pregnancy. VPA treatment also induces autistic-like behaviors in the offspring of rats after prenatal exposure; hence it is a preclinical disease model with high translational value. In the present study, our aim was to characterize ASD relevant behaviors of socially housed, individually identified male rats in automated home cages. The natural behavior of rats was assessed by monitoring their visits to drinking bottles in an environment without human influence aiming at reducing interventional stress. Although rodents normally tend to explore their new environment, prenatally VPA-treated rats showed a drastic impairment in initial and long-term exploratory behavior throughout their stay in the automated cage. Furthermore, VPA rats displayed psychogenic polydipsia (PPD) as well as altered circadian activity. In the competitive situation of strict water deprivation controls switched to an uneven resource sharing and only a few dominant animals had access to water. In VPA animals similar hierarchy-related changes were completely absent. While the control rats secured their chance to drink with frequent reentering visits, thereby "guarding" the water resource, VPA animals did not switch to uneven sharing and displayed no evidence of guarding behavior.

Purkinje cell number-correlated cerebrocerebellar circuit anomaly in the valproate model of autism.

While cerebellar alterations may play a crucial role in the development of core autism spectrum disorder (ASD) symptoms, their pathophysiology on the function of cerebrocerebellar circuit loops is largely unknown. We combined multimodal MRI (9.4 T) brain assessment of the prenatal rat valproate (VPA) model and correlated immunohistological analysis of the cerebellar Purkinje cell number to address this question. We hypothesized that a suitable functional MRI (fMRI) paradigm might show some altered activity related to disrupted cerebrocerebellar information processing. Two doses of maternal VPA (400 and 600 mg/kg, s.c.) were used. The higher VPA dose induced 3% smaller whole brain volume, the lower dose induced 2% smaller whole brain volume and additionally a focal gray matter density decrease in the cerebellum and brainstem. Increased cortical BOLD responses to whisker stimulation were detected in both VPA groups, but it was more pronounced and extended to cerebellar regions in the 400 mg/kg VPA group. Immunohistological analysis revealed a decreased number of Purkinje cells in both VPA groups. In a detailed analysis, we revealed that the Purkinje cell number interacts with the cerebral BOLD response distinctively in the two VPA groups that highlights atypical function of the cerebrocerebellar circuit loops with potential translational value as an ASD biomarker.

P2Y receptor mediated inhibitory modulation of noradrenaline release in response to electrical field stimulation and ischemic conditions in superfused rat hippocampus slices.

In this study, the inhibitory regulation of the release of noradrenaline (NA) by P2 receptors was investigated in hippocampus slices pre-incubated with [(3)H]NA. Electrical field stimulation (EFS; 2 Hz, 240 shocks, and 1 ms) released NA in an outside [Ca(2+)]-dependent manner, and agonists of P2Y receptors inhibited the EFS-evoked [(3)H]NA release with pharmacological profile similar to that of the P2Y(1) and P2Y(13) receptor subtypes. This inhibitory modulation was counteracted by bicuculline and 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline + 2-amino-5-phosphonovalerate and 2-amino-4-phosphonobutyrate. In contrast, the excess release in response to 30 min combined oxygen and glucose deprivation was outside [Ca(2+)] independent, but still sensitive to the inhibition of both facilitatory P2X(1) and inhibitory P2Y(1) receptors. Whereas mRNA encoding P2Y(12) and P2Y(13) receptor subunits were expressed in the brainstem, P2Y(1) receptor immunoreactivity was localized to neuronal somata and dendrites innervated by the mossy fiber terminals in the CA3 region of the hippocampus, as well as somata of granule cells and interneurons in the dentate gyrus. In summary, in addition to the known facilitatory modulation via P2X receptors, EFS-evoked [(3)H]NA outflow in the hippocampus is subject to inhibitory modulation by P2Y(1)/P2Y(13) receptors. Furthermore, endogenous activation of both facilitatory and inhibitory P2 receptors may participate in the modulation of pathological NA release under ischemic-like conditions.

Modulation of neurotransmitter release by P2X and P2Y receptors in the rat spinal cord.

In this study, the P2 receptor-mediated modulation of [3H]glutamate and [3H]noradrenaline release were examined in rat spinal cord slices. Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and 2-methylthioadenosine 5'-diphosphate (2-MeSADP) decreased the electrical stimulation-evoked [3H]glutamate efflux with the following order of potency: ADP>2-MeSADP>ATP. The effect of ATP was antagonized by suramin (300microM), the P2Y12,13 receptor antagonist 2-methylthioadenosine 5'-monophosphate (2-MeSAMP, 10microM), and partly by 4-[[4-Formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS, 30microM) and the P2Y1 receptor antagonist 2'-deoxy-N6-methyladenosine 3',5'-diphosphate (MRS 2179, 10muM). ATP, ADP and 2-MeSADP also decreased evoked [3H]noradrenaline outflow; the order of agonist potency was ADP> or =2-MeSADP>ATP. The effect of ATP was reversed by 2-MeSAMP (10microM), and partly by MRS 2179 (10microM). By contrast, 2-methylthioadenosine-5'-triphosphate (2-MeSATP, 10-300microM) increased resting and electrically evoked [3H]glutamate and [3H]noradrenaline efflux, and this effect was prevented by the P2X1 receptor selective antagonist 4,4',4'',4'''-[carbonylbis[imino-5,1,3-benzenetriyl bis (carbonyl-imino)]] tetrakis (benzene-1,3-disulfonic acid) octasodium salt (NF449, 100nM). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that mRNAs encoding P2Y12 and P2Y13 receptors are expressed in the brainstem, whereas P2Y13 but not P2Y12 receptor mRNA is present in the dorsal root ganglion and spinal cord. P2Y1 receptor expression in the spinal cord is also demonstrated at the protein level. In conclusion, inhibitory P2Y and facilitatory P2X1-like receptors, involved in the regulation of glutamate (P2Y13 and/or P2Y1) and noradrenaline (P2Y13 and/or P2Y1, P2Y12) release have been identified, which provide novel target sites for analgesics acting at the spinal cord level.

The role of P2X7 receptors in a rodent PCP-induced schizophrenia model.

P2X7 receptors (P2X7Rs) are ligand-gated ion channels sensitive to extracellular ATP. Here we examined for the first time the role of P2X7R in an animal model of schizophrenia. Using the PCP induced schizophrenia model we show that both genetic deletion and pharmacological inhibition of P2X7Rs alleviate schizophrenia-like behavioral alterations. In P2rx7+/+ mice, PCP induced hyperlocomotion, stereotype behavior, ataxia and social withdrawal. In P2X7 receptor deficient mice (P2rx7-/-), the social interactions were increased, whereas the PCP induced hyperlocomotion and stereotype behavior were alleviated. The selective P2X7 receptor antagonist JNJ-47965567 partly replicated the effect of gene deficiency on PCP-induced behavioral changes and counteracted PCP-induced social withdrawal. We also show that PCP treatment upregulates and increases the functional responsiveness of P2X7Rs in the prefrontal cortex of young adult animals. The amplitude of NMDA evoked currents recorded from layer V pyramidal neurons of cortical slices were slightly decreased by both genetic deletion of P2rx7 and by JNJ-47965567. PCP induced alterations in mRNA expression encoding schizophrenia-related genes, such as NR2A, NR2B, neuregulin 1, NR1 and GABA α1 subunit were absent in the PFC of young adult P2rx7-/- animals. Our findings point to P2X7R as a potential therapeutic target in schizophrenia.

Neurochemical Changes in the Mouse Hippocampus Underlying the Antidepressant Effect of Genetic Deletion of P2X7 Receptors.

Recent investigations have revealed that the genetic deletion of P2X7 receptors (P2rx7) results in an antidepressant phenotype in mice. However, the link between the deficiency of P2rx7 and changes in behavior has not yet been explored. In the present study, we studied the effect of genetic deletion of P2rx7 on neurochemical changes in the hippocampus that might underlie the antidepressant phenotype. P2X7 receptor deficient mice (P2rx7-/-) displayed decreased immobility in the tail suspension test (TST) and an attenuated anhedonia response in the sucrose preference test (SPT) following bacterial endotoxin (LPS) challenge. The attenuated anhedonia was reproduced through systemic treatments with P2rx7 antagonists. The activation of P2rx7 resulted in the concentration-dependent release of [(3)H]glutamate in P2rx7+/+ but not P2rx7-/- mice, and the NR2B subunit mRNA and protein was upregulated in the hippocampus of P2rx7-/- mice. The brain-derived neurotrophic factor (BDNF) expression was higher in saline but not LPS-treated P2rx7-/- mice; the P2rx7 antagonist Brilliant blue G elevated and the P2rx7 agonist benzoylbenzoyl ATP (BzATP) reduced BDNF level. This effect was dependent on the activation of NMDA and non-NMDA receptors but not on Group I metabotropic glutamate receptors (mGluR1,5). An increased 5-bromo-2-deoxyuridine (BrdU) incorporation was also observed in the dentate gyrus derived from P2rx7-/- mice. Basal level of 5-HT was increased, whereas the 5HIAA/5-HT ratio was lower in the hippocampus of P2rx7-/- mice, which accompanied the increased uptake of [(3)H]5-HT and an elevated number of [(3)H]citalopram binding sites. The LPS-induced elevation of 5-HT level was absent in P2rx7-/- mice. In conclusion there are several potential mechanisms for the antidepressant phenotype of P2rx7-/- mice, such as the absence of P2rx7-mediated glutamate release, elevated basal BDNF production, enhanced neurogenesis and increased 5-HT bioavailability in the hippocampus.

The inhibitory action of exo- and endocannabinoids on [³H]GABA release are mediated by both CB1and CB2receptors in the mouse hippocampus.

Exogenous and endogenous cannabinoids play an important role in modulating the release of neurotransmitters in hippocampal excitatory and inhibitory networks, thus having profound effect on higher cognitive and emotional functions such as learning and memory. In this study we have studied the effect of cannabinoid agonists on the potassium depolarization-evoked [(3)H]GABA release from hippocampal synaptosomes in the wild-type (WT) and cannabinoid 1 receptor (CB(1)R)-null mutant mice. All tested cannabinoid agonists (WIN55,212-2, CP55,940, HU-210, 2-arachidonoyl-glycerol, 2-AG; delta-9-tetra-hydrocannabinol, THC) inhibited [(3)H]GABA release in WT mice with the following rank order of agonist potency: HU-210>CP55,490>WIN55,212-2>>2-AG>THC. By contrast, 2-AG and THC displayed the greatest efficacy eliciting almost complete inhibition of evoked [(3)H]GABA efflux, whereas the maximal inhibition obtained by HU-210, CP55,490, and WIN55,212-2 were less, eliciting not more than 40% inhibition. The inhibitory effect of WIN55,212-2, THC and 2-AG on evoked [(3)H]GABA efflux was antagonized by the CB(1) receptor inverse agonist AM251 (0.5 µM) in the WT mice. In the CB(1)R knockout mice the inhibitory effects of all three agonists were attenuated. In these mice, AM251 did not antagonize, but further reduced the [(3)H]GABA release in the presence of the synthetic agonist WIN55,212-2. By contrast, the concentration-dependent inhibitory effects of THC and 2-AG were partially antagonized by AM251 in the absence of CB(1) receptors. Finally, the inhibition of evoked [(3)H]GABA efflux by THC and 2-AG was also partially attenuated by AM630 (1 µM), the CB(2) receptor-selective antagonist, both in WT and CB(1) knockout mice. Our data prove the involvement of CB(1) receptors in the effect of exo- and endocannabinoids on GABA efflux from hippocampal nerve terminals. In addition, in the effect of the exocannabinoid THC and the endocannabinoid 2-AG, non-CB(1), probably CB(2)-like receptors are also involved.

Lack of neuroprotection in the absence of P2X7 receptors in toxin-induced animal models of Parkinson's disease.

BACKGROUND: Previous studies indicate a role of P2X7 receptors in processes that lead to neuronal death. The main objective of our study was to examine whether genetic deletion or pharmacological blockade of P2X7 receptors influenced dopaminergic cell death in various models of Parkinson's disease (PD). RESULTS: mRNA encoding P2X7 and P2X4 receptors was up-regulated after treatment of PC12 cells with 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). P2X7 antagonists protected against MPTP and rotenone induced toxicity in the LDH assay, but failed to protect after rotenone treatment in the MTT assay in PC12 cells and in primary midbrain culture. In vivo MPTP and in vitro rotenone pretreatments increased the mRNA expression of P2X7 receptors in the striatum and substantia nigra of wild-type mice. Basal mRNA expression of P2X4 receptors was higher in P2X7 knockout mice and was further up-regulated by MPTP treatment. Genetic deletion or pharmacological inhibition of P2X7 receptors did not change survival rate or depletion of striatal endogenous dopamine (DA) content after in vivo MPTP or in vitro rotenone treatment. However, depletion of norepinephrine was significant after MPTP treatment only in P2X7 knockout mice. The basal ATP content was higher in the substantia nigra of wild-type mice, but the ADP level was lower. Rotenone treatment elicited a similar reduction in ATP content in the substantia nigra of both genotypes, whereas reduction of ATP was more pronounced after rotenone treatment in striatal slices of P2X7 deficient mice. Although the endogenous amino acid content remained unchanged, the level of the endocannabinoid, 2-AG, was elevated by rotenone in the striatum of wild-type mice, an effect that was absent in mice deficient in P2X7 receptors. CONCLUSIONS: We conclude that P2X7 receptor deficiency or inhibition does not support the survival of dopaminergic neurons in an in vivo or in vitro models of PD.

P2 receptor-mediated modulation of neurotransmitter release-an update.

Presynaptic nerve terminals are equipped with a number of presynaptic auto- and heteroreceptors, including ionotropic P2X and metabotropic P2Y receptors. P2 receptors serve as modulation sites of transmitter release by ATP and other nucleotides released by neuronal activity and pathological signals. A wide variety of P2X and P2Y receptors expressed at pre- and postsynaptic sites as well as in glial cells are involved directly or indirectly in the modulation of neurotransmitter release. Nucleotides are released from synaptic and nonsynaptic sites throughout the nervous system and might reach concentrations high enough to activate these receptors. By providing a fine-tuning mechanism these receptors also offer attractive sites for pharmacotherapy in nervous system diseases. Here we review the rapidly emerging data on the modulation of transmitter release by facilitatory and inhibitory P2 receptors and the receptor subtypes involved in these interactions.