Rómeó D. Andó

A comparative analysis of the activity of ligands acting at P2X and P2Y receptor subtypes in models of neuropathic, acute and inflammatory pain

Background and purpose:  This study was undertaken to compare the analgesic activity of antagonists acting at P2X1, P2X7, and P2Y12 receptors and agonists acting at P2Y1, P2Y2, P2Y4, and P2Y6 receptors in neuropathic, acute, and inflammatory pain.

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.

K+ depolarization evokes ATP, adenosine and glutamate release from glia in rat hippocampus: a microelectrode biosensor study

BACKGROUND AND PURPOSE This study was undertaken to characterize the ATP, adenosine and glutamate outflow evoked by depolarization with high K+ concentrations, in slices of rat hippocampus.

Neurochemical evidence that stimulation of CB1 cannabinoid receptors on GABAergic nerve terminals activates the dopaminergic reward system by increasing dopamine release in the rat nucleus accumbens.

We examined the effect of cannabinoid receptor activation on basal and electrical field simulation-evoked (25 V, 2 Hz, 240 shocks) [(3)H]dopamine efflux in the isolated rat nucleus accumbens in a preparation, in which any effect on the dendrites or somata of ventral tegmental projection neurons was excluded. The cannabinoid agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN55,212-2, 100 nM) significantly enhanced stimulation-evoked [(3)H]dopamine release in the presence of the selective dopamine transporter inhibitor 1-[2-[bis-(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine dihydrochloride (GBR12909, 100 nM). GBR12909 (100 nM-1 microM), when added alone, increased the evoked [(3)H]dopamine efflux in a concentration-dependent manner. The stimulatory effect of WIN55,212-2 on the evoked tritium efflux was inhibited by the selective CB1 cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251, 100 nM) and by the GABA(A) receptor antagonist bicuculline (10 microM). Repeated application of N-methyl-d aspartate (1 mM) under Mg(2+)-free conditions, which directly acts on dopaminergic terminals, reversibly increased the tritium efflux, but WIN55,212-2 did not affect N-methyl-d aspartate-evoked [(3)H]dopamine efflux, indicating that WIN55,212-2 has no direct action on dopaminergic nerve terminals. AM251 (100 nM) alone also did not have an effect on electrical stimulation-evoked [(3)H]dopamine efflux. Likewise, the selective CB2 receptor antagonist 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630, 0.3 microM) and the anandamide transport inhibitor (5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide (VDM11, 10 microM) had no significant effect on electrically evoked [(3)H]dopamine release. This is the first neurochemical evidence that the activation of CB1 cannabinoid receptors leads to the augmentation of [(3)H]dopamine efflux via a local GABA(A) receptor-mediated disinhibitory mechanism in the rat nucleus accumbens.

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

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-1β 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 (0.3 mg/kg i.t.) attenuated CFA-induced expression of cytokines in the hind paw and of IL-1β 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-1β. 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 inhibitory action of exo- and endocannabinoids on [ 3H]GABA release are mediated by both CB 1 and CB 2 receptors 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.

The effects of optical, electrical and chemical stimulation on serotonin release from median raphe and hippocampus of mice

The present study has examined several characteristics of the release of [3H]5-HT from the median raphe nucleus (MRN) and hippocampus in terms of its dependence of nerve impulse. We used electrical stimulation and the sodium channel opener veratridine, which excite all of the neuronal processes in the stimulation field, and optogenetics to selectively stimulate those terminals which express channelrhodopsin-2 (ChR2) and compared 5-HT release evoked by electrical and chemical depolarization and by light. We injected an adeno-associated virus containing DNA construct encoding ChR2 into the MRN of mice and investigated [3H]5-HT release from MRN and hippocampal slices. Serotonergic nerve terminals was locally stimulated with 473 nm light (blue laser diode) and electrically by bipolar electrode and by veratridine and transmitter release was monitored by collecting the effluent in a fraction collector. Electrical filed stimulation and veratridine resulted in a significantly increase in the efflux of 5-HT, whereas optical stimulation of ChR2 expressing nerve terminals at various frequencies (10, 20, 50, 100 Hz) elicited only a negligible increase in 5-HT release either from the hippocampus or from the MRN itself. The electrically induced release of radioactive neurotransmitter was completely inhibited by perfusion with tetrodotoxin. We have also applied the 5-HT transporter inhibitor, fluoxetine and the GABAA blocker bicuculline to relieve released 5-HT from re-uptake and any endogenous inhibition. Nevertheless, the effect of optical stimulation remained closed to the detection limit under these condition. In conclusion, whereas our method is suitable to detect [3H]5-HT efflux in response to ongoing neuronal sodium channel activity its sensitivity is too low to detect transmitter efflux evoked by focal optogenetic stimulation. The most likely reason for the failure of detection of 5-HT efflux is that ChR2 is expressed only by a small subpopulation of nerve terminals.

The role of P2X7 ATP receptors in the nervous system: potential implications in inflammatory and depression-like diseases

Background The P2X7 receptor is a ligand-gated ion channel expressed in neuronal, glial and immune cells and is implicated in a wide range of pathological conditions, including ischemia, and inflammation. The P2X7 receptor can modulate the maturation and release of the proinflammatory cytokine, interleukin-1β (IL-1β). IL-1β is suggested to be involved in the pathophysiology of depression and sickness behaviour, elicited by peripherally administered bacterial lipopolysaccharide (LPS).